Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone.

Gibbons (Family Hylobatidae) are a suitable model for exploring hybridization in pair-living primates as several species form hybrid zones. In Khao Yai National Park, Thailand, white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) are distributed parapatrically and hybridize in a narrow zone. Their phenotypic characteristics suggest limited inter-species gene flow, although this has never been assessed. To uncover the history and degree of gene flow between the two species, we studied the genetic structure of gibbons in the hybrid zone by analyzing fecal DNA samples, phenotypic characteristics, vocalizations and individuals' social status. We determined eight autosomal single nucleotide variant (SNV) loci, and mitochondrial DNA (mtDNA) and Y-chromosomal haplotypes of 72 gibbons. We compared these markers with reference types of wild pureblood white-handed gibbons (n = 12) in Kaeng Krachan National Park and pureblood pileated gibbons (n = 4) in Khao Soi Dao Wildlife Sanctuary. Autosomal genotypic analyses confirmed the various levels of mixed ancestry for several adult gibbons with or without atypical phenotypic traits in Khao Yai National Park. In some other adult gibbons, the mixed ancestry was not detected in either autosomal SNVs or their phenotypic traits but the mtDNA. Both male and female adult hybrids formed reproductive units mainly with a phenotypic pureblood partner and many of them produced offspring. Taken together, our results suggest that once hybridization occurs, white-handed-pileated-gibbon hybrids can reproduce with either parental species and that the backcrossing and thus introgression may occur in successive generations, with no drastic changes in phenotypic appearance.

Introduction

Natural hybridization and introgression are widespread phenomena in animals [1], including primates [2, 3]. Genome-scale studies have revealed “hidden” ancient (or historical) hybridization and introgression among many primate taxa (e.g. chimpanzees and bonobos [4], orangutans [5], baboons [6] and macaques [7]). While genome-scale studies are suitable for uncovering the historical impact of hybridization and introgression in primate evolution, field observations in hybrid zones are essential to investigate proximate mechanisms that prevent (i.e., produce reproductive barriers) or promote hybridization [8]. Integrating field- with lab-based studies can help to illuminate entire hybridization/introgression histories [9]. For example, baboons are one major model of studying hybridization in primates because a long history of field observations [10], genomic studies [6] and an integration of both exists [11, 12].

Gibbons (family Hylobatidae) provide an equally interesting model for studying hybridization. First, among extant apes, only gibbons show ongoing, active hybrid zones [13-15]. Second, their typical reproductive system is monogamous pairs, and they reproduce primarily with the pair-living partner [16, 17], although some variations in social organizations [18-20], mating patterns [19, 21–23] and reproductive patterns [16, 24] have also been observed. Wild gibbons start reproduction at about 10 years of age, and an adult female reproduces only once in about every three years [25]. Considering the low ratio of extra-pair paternity [16, 17, 24], both female and male gibbons reproduce a limited number of offspring into which they invest substantially. The monogamous reproductive pattern seems more sensitive to potential disadvantages of hybridization [26], such as, for example, testicular dysfunction and sterility in hybrid males [27], compared to polygamous and multiparous reproductive system. Finally, gibbon species share basic biological characteristics, such as being adapted to a mainly frugivorous diet and life in the canopy [28]. Competitive inter-species relationships have been suggested for most species [29], which is indirectly supported by the notion of an allopatric distribution of species, except for the sympatric distribution between large-bodied siamangs (Symphalangus syndactylus) and smaller gibbons of the genus Hylobates [30]. Species differences in gibbons appear in their vocal repertoire and pelage coloration [15], which are important in social communication and probably as well in species identification.

Hybridization between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in Khao Yai National Park, Thailand, has been studied for more than 40 years [13, 15, 31–33]. Observations have uncovered a limited number of hybrid gibbons that have been identified by mixed-species vocalizations and external morphology, such as pelage coloration, distributed in a narrow zone along the Lam Ta Khong River [13]. Early studies have argued that the hybrid population could be a “sink population” where introgression (or gene flow) between the two species is limited [13, 33]. However, since sophisticated molecular methods were unavailable in the 1970s and 1980s, the argument of an existing “sink hybrid population” could not be proven at the molecular level. Recently, we analyzed mtDNA of a population that was thought of as “pure” white-handed gibbons, at the Mo Singto study site in Khao Yai, only to find confirmation that some phenotypically white-handed gibbons possessed mtDNA of a typical pileated gibbon haplotype [34]. This characteristic suggested the presence of several backcross generations and some introgression between the two species even within the Mo Singto subpopulation of phenotypic white-handed gibbons. Moreover, another recent study analyzing genome-wide single nucleotide variants (SNVs) among captive gibbons detected an introgression signal between every combination of the two species individuals (among 20 white-handed gibbons and 10 pileated gibbons), indicative of a relatively long hybridization history [35].

To fully understand the pattern of hybridization in a long-established hybrid zone, the present study aimed to uncover the degree of introgression and mating patterns between white-handed and pileated gibbons in the ongoing, active natural hybrid zone in Khao Yai National Park, Thailand, one of the oldest National Parks in Thailand which since it’s establishment in 1962 has had no meaningful connectivity to other forests in Thailand. We analyzed autosomal SNVs, together with mtDNA and Y-chromosome polymorphisms among gibbons living in the natural hybrid zone and assessed the presence of introgression by analyzing the admixture level of individuals and morphological characteristics. We also compared hybrids with pureblood white-handed gibbons in Kaeng Krachan National Park (approximately 250 km Southwest of the Khao Yai hybrid zone) and pureblood pileated gibbons in Khao Soi Dao Wildlife Sanctuary (approximately 150 km Southeast of the Khao Yai hybrid zone; Fig 1). Based on the genetic analyses and observations, we assessed hybrid gibbons’ mating patterns to uncover their reproductivity and direction of introgression, which is potentially biased between the two species.

Fig 1

Distribution of white-handed gibbons (Hylobates lar), pileated gibbons (H. pileatus) and study sites.

The approximate species distribution range was manually drawn based on previous studies [15, 36] and the Gibbon Research Lab (http://www.gibbons.de/main2/). The protected areas, country borders, coastlines and rivers came from OpenStreetMap and OpenStreetMap Foundation, which was available under the Open Database License.

Methods

Ethical statement

We conducted the study under the permission of the National Research Council of Thailand (NRCT); the Department of National Parks, Wildlife and Plant Conservation of Thailand (DNP); Khao Yai National Park; Kaeng Krachan National Park; and Khao Soi Dao Wildlife Sanctuary (NRCT project No. 2757).

Study sites and distribution survey

We conducted a field survey in the natural hybrid zone of white-handed gibbons and pileated gibbons in Khao Yai National Park, Thailand (N 14.43872°, E 101.37238°) (Fig 1), from February to December 2014. In this study, we selected three survey areas: (1) Mo Singto, (2) the western side of the valley of Lam Ta Khong River and (3) the northern slope of Khao Khiao (Fig 2), where the high potential of the presence of hybrid gibbons was expected based on previous studies and accessibility.

Fig 2

Three study areas in Khao Yai National Park.

Black solid lines in the main map represent motorways. Light blue lines represent rivers. Light green areas represent grasslands. The border of Khao Yai National Park, roads, rivers and grasslands came from OpenStreetMap and OpenStreetMap Foundation, which was available under the Open Database License.

We estimated the presence of gibbon groups and their putative species identity by their prominent vocalization [37]. We set up 21 listening posts each about 500 m apart from one another along nature trails and motorways. We conducted initial listening survey from only one listening post at a time, spending one to three days at each listening post based on listening conditions. When we noticed gibbons’ vocalizations, we recorded start and end times, song category (i.e. duets, OA duets, disturbance-induced songs, male solos and others), the songs’ putative species identity, and approximate distance and direction from the listening post to the singing location. In addition to listening post surveys, we also recorded the same gibbon vocalization parameters whenever we were in the forest.

After the initial listening survey, we also directly followed and observed gibbon groups, and collected fecal samples. Whenever we encountered gibbons, we recorded the start and end times of direct observation. We obtained encounter locations from a GPS data logger (GPSMAP60CSx, Garmin, KS) brought by the observer. GPS coordinates were extracted in 5 min intervals and plotted on a map to estimate gibbon groups’ home ranges.

We did not know for how long hybridization had occurred in Khao Yai. Thus, we suspected that many white-handed and pileated gibbons’ morphospecies in Khao Yai might have some amount of mixed genetic background. Therefore, Khao Yai gibbons could not be considered good comparative sample to represent pureblood white-handed or pureblood pileated gibbons. To avoid this problem in our genetic analysis of identifying hybrids, we selected our comparative samples from populations more than a hundred kilometers distant to the hybrid zone, from deep within each species’ distribution range, and conducted field surveys for white-handed gibbons in Kaeng Krachan National Park (12.88499° N, 99.63266° E) from March to April 2015, and for pileated gibbons in Khao Soi Dao Wildlife Sanctuary (13.10296° N, 102.19195° E) from February to April 2016 (Fig 1). These study sites were about 180 km and 130 km linear distance apart, respectively, from each species’ closest putative historical species contact zone [38]. Based on the distance far from the hybrid zone and the morphospecies, effects of recent hybridization between the two species were considered negligible.

Group composition and morphological data collection

When we encountered a gibbon group, we identified each group member’s sex and all members’ age category: adult (>8 years old and the main adult member), subadult (6–8 years old, >8 years old and not the main adult member), adolescent (4–6 years old), juvenile (2–4 years old) and infant (0–2 years old) (modified after [39]). We recorded the gibbons’ morphological characteristics using a digital camera (Lumix FZ-200, Panasonic, Japan).

DNA sample collection, extraction and quantification

Fecal samples were collected as a source of DNA. We used the well established ethanol–silica two-step method [40]. We kept samples at room temperature at the study sites for up to one year and later stored them at 4°C in a lab refrigerator until they were processed. We also collected some fecal samples in lysis solution by using cotton buds [41]. We collected 129 fecal samples in Khao Yai, 71 fecal samples in Kaeng Krachan, and nine fecal samples in Khao Soi Dao. Because we expected that not every fecal sample would contain sufficient amounts of DNA for genotyping, multiple samples were collected from the same individuals to ensure success in genotyping. Fecal sample collection from non-habituated gibbons was difficult and the number of samples collected for each individual remained limited. Furthermore, we collected hundreads of fecal samples in Khao Yain National Park in previous studies [16, 34]. Thus, we also selected and genotyped DNA samples of adult gibbons from the previous samples. Overall, 88 individuals were genotyped. Of those, 65 adult plus seven non-adult gibbons were genotyped from Khao Yai, 12 adult gibbons were genotyped from Kaeng Krachan and four adult gibbons were genotyped from Khao Soi Dao.

We extracted DNA using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) with some modifications. About 200 mg feces (or 200 mL lysis buffer) were vortex-mixed with 1 mL InhibitEX® Buffer and incubated overnight at room temperature, shaking with a tube mixer. We extended the incubation time with proteinase K for 1 h. Furthermore, the final incubation step with 200 μL elution buffer (Buffer ATE) lasted 30 min.

DNA extracted from fecal samples included host and non-host DNA, such as gastrointestinal microbiota. Since the amount of host DNA was critical for the DNA analysis [42], we thus measured the concentration of host DNA using quantitative real-time PCR targeting c-myc [42, 43] for quality control. The reaction was conducted in the same way as in a previous study [16].

SNV genotyping

We searched for SNVs using two approaches. First, the DNA sequences of autosomal noncoding loci [44] were obtained from GenBank. The data set consisted of 11 white-handed and four pileated gibbons. We selected SNV sites fixed between the two species, and no other SNVs were detected at least 45 bases upstream and downstream of the target SNV sites, where primers and probes were designed. In this process, 14 loci were available from the database and seven of those loci fulfilled the above criteria. Of those, two loci were estimated to be located on the close position at a chromosome, and thus one of them was excluded and six loci were tested for genotyping. Genotyping succeeded in five of the six loci. Second, we also screened autosomal SNV loci by using primers reported by Perelman et al. [45], who determined a sequence of one white-handed gibbon, one agile gibbon (Hylobates agilis), one Müller’s gibbon (Hylobates muelleri) and one siamang. The sequence of 39 autosomal loci was available for the white-handed gibbon sample. Of those, 17 loci held SNVs which were specific to the white-handed gibbon sample and different from the other three gibbon species samples and the genome sequence of a northern white-cheeked gibbon (Nomascus leucogenys), nomLeu3/Nleu_3.0 [46]. In 12 of the 17 loci, SNVs were located in non-coding regions. We determined the sequences of five of the 12 loci in white-handed and pileated gibbon DNA samples from our DNA repository by Sanger sequencing and three loci held suitable SNV sites for designing primers and probes. In both the first and the second approaches, to avoid genotyping linked loci, we checked the approximate location of each SNV in the white-handed gibbon genome by comparing it with the northern white-cheeked gibbon genome sequence [46] and the result of chromosome painting [47]. Based on the sequences, TaqMan probes and primers for SNV loci were designed by Applied Biosystems (MA, United States) (S1 Table).

We genotyped eight SNVs by real-time PCR using TaqMan MGB Probes. Each 10 μL reaction mix contained 1× TaqMan GTXpress Master Mix (Applied Biosystems), 1× TaqMan SNP Genotyping Assay (Applied Biosystems) and 2 μL of DNA template. PCR cycle conditions were as follows: preincubation at 25°C for 30 s and at 95°C for 20 s, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. We conducted the reactions with a StepOnePlus Real-Time PCR System (Applied Biosystems) or a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). We initially duplicated genotyping of a subset of DNA samples for three SNV loci and obtained the same genotypes for each repetition, except when genotyping failed. This indicates that allelic dropout in our real-time PCR condition was negligible, and thus we conducted only one reaction per locus per sample for further genotyping.

Ancestry analysis

We estimated each individual’s genetic admixture levels by STRUCTURE Version 2.3.4 [48]. We used the admixture model of Pritchard et al. [48] with the number of populations (K), from 2 to 5. We performed 10 independent runs for each K. Each run consisted of 1,000,000 generations of Markov chain Monte Carlo with a burn-in period of 100,000 generations. Proportions of each ancestry for each animal were averaged over the 10 independent runs.

Mitochondrial DNA (mtDNA) and Y-chromosome genotyping

We determined the haplotypes of hypervariable region 1 (HVR1) of the mtDNA (631- or 632-base fragment) and the Y-chromosome haplogroups based on an sry indel (2-base indel), as done in previous studies [34, 49]. Based on the 2-base indel in the non-coding region of the sry gene, it was possible to distinguish two substantially different sequence groups, which we named haplogroup YA (2 bases longer) and YB (2 bases shorter) [49]. In the study [49], only YA was observed among captive white-handed gibbons, but both haplogroups YA and YB were observed among captive pileated gibbons. The same pattern was reported in another independent study that focused on different regions of the Y-chromosome [50]. We outsourced the sequencing and fragment analyses of PCR products to Macrogen (Korea). The electropherograms of HVR1 were visualized by MEGA6 [51] and each sequence was determined. We made a median-joining network [52] of HVR1 by using PopART 1.7 [53], and assigned haplotypes based on the network drawn. We determined the sry PCR product’s fragment size using GeneMapper (Applied Biosystems) and assigned haplogroups based on a previous study [49].

Results

Mixed ancestry of autosomes in Khao Yai gibbons

The STRUCTURE analysis of the eight autosomal SNVs successfully distinguished white-handed and pileated gibbon ancestry from one another when K was 2 (Fig 3C). When K was 3 to 5, the white-handed gibbon ancestry of each individual was almost evenly divided into 2 to 4 components, and the pileated gibbon ancestry was not changed across the different K values. Thus, we considered that the genetic makeup of the gibbon individuals was well represented by result when K = 2. Adult gibbons in Kaeng Krachan (n = 11 of 12) showed 0.995–0.996 for white-handed ancestry and 0.004–0.005 for pileated ancestry, while the remaining individual showed marginally lower respective higher values of 0.983 for white-handed and 0.017 for pileated ancestry. On the other hand, adult gibbons in Khao Soi Dao (n = 4) showed 0.004 for white-handed and 0.996 for pileated ancestry.

Fig 3

White-handed and pileated gibbon ancestry of gibbons in Kaeng Krachan, Khao Yai and Khao Soi Dao.

(A) Y-chromosome. Yellow: haplogroup YA; orange: haplogroup YB. (B) mtDNA and (C) autosome. Green: white-handed gibbon ancestry; purple: pileated gibbon ancestry. Black triangles represent intermediate hybrid gibbons. Blank triangles represent hybrid gibbon with low levels of mixed ancestry. Blank circles represent “possible hybrid” gibbons with marginal mixed ancestry. M: male; F: female; U: sex unidentified.

In Khao Yai, levels of mixed ancestry were varied. Among 65 adult gibbons, 18 showed a mixed ancestry of white-handed (0.392 to 0.986) and pileated (0.014 to 0.608) gibbons (Fig 3C). Of those, five (three females, two males) showed intermediate values, approximately a half white-handed (0.392 to 0.527) and half pileated (0.473 to 0.608) ancestry, and five adult gibbons (four females, one male) showed stronger biased ancestry with low levels of pileated ancestry (0.022 to 0.185). We considered those ten gibbons as hybrid gibbons. The other eight gibbons (one female, seven males) with low levels of marginal pileated ancestry (0.014 to 0.018) but non-negligible levels of mixed ancestry (0.004 to 0.005), were considered “possible hybrid” gibbons. We based our decision on the fact that one pureblood white-handed gibbon in Kaeng Krachan showed pileated ancestry at 0.017, perhaps due to the small number of SNV loci used in the analysis and an incomplete segregation of SNV markers. Thus, a marginal value around 0.017 could be found in both hybrid and pureblood gibbons. Among seven non-adult gibbons observed in groups consisting intermediate hybrid adults, six showed mixed ancestry, with values of pileated ancestry ranging from 0.255 to 0.607. Another 42 adults and one non-adult gibbon showed quite low levels of pileated ancestry (0.004 to 0.005) at similar levels observed also among pureblood white-handed gibbons in Kaeng Krachan, and thus these individuals were identified as white-handed gibbons. Five adult gibbons showed large pileated ancestry (0.994 to 0.996) and almost no mixed ancestry of white-handed gibbons (0.004 to 0.006) at the same levels also observed among pureblood pileated gibbons in Khao Soi Dao, and thus these individuals were identified as pileated gibbons.

Mitochondrial DNA and Y-chromosome ancestry

We found 32 haplotypes of HVR1 of mtDNA among 88 gibbons from the three study sites (S2 Table). Of those haplotypes, 21 newly found ones were deposited in DDBJ/EMBL/GenBank (Accession No. LC633853–LC633873). White-handed gibbons at Kaeng Krachan (n = 12) and pileated gibbons at Khao Soi Dao (n = 4) showed eight white-handed and three pileated typical mtDNA haplotypes. None of them were shared by Khao Yai gibbons (S1 Fig).

In Khao Yai, among ten adult hybrid gibbons, eight showed white-handed mtDNA haplotypes, and two showed the pileated mtDNA haplotype (Fig 3). All eight adult “possible hybrid” gibbons showed white-handed mtDNA haplotypes. Among 42 adult gibbons who were identified as white-handed gibbons based on the eight autosomal SNV loci, four shared a single pileated mtDNA haplotype (HKY11B) and the others showed white-handed mtDNA haplotypes. Conversely, among five adult gibbons who identified as pileated gibbons based on the eight autosomal SNVs, all showed only pileated mtDNA haplotypes. MtDNA haplotype of non-adult hybrid gibbons were identical to that of the adult hybrid female in the same group (HKY18B in AA1; HKY16A in AC4; HKY15A in AC3), suggesting mother-offspring relationships.

In the Y-chromosome, among Kaeng Krachan subjects, seven adult male white-handed gibbons showed only haplogroup YA. Among Khao Soi Dao subjects, we failed to amplify the Y-chromosome from the only pilated gibbon male in the sample. Among Khao Yai subjects, 26 adult male white-handed gibbons showed haplogroup YA, while three adult male pileated gibbons showed haplogroups YA (n = 2) and YB (n = 1), similar to previous results in captive gibbons [49, 50] (Fig 3). Because of the lack of Y-chromosome data for pileated gibbons at Khao Soi Dao, it was not possible to conclude whether or not the two haplogroups in pileated gibbons at Khao Yai occurred because of recent introgression or a natural polymorphism (by incomplete lineage sorting or ancient introgression). The three adult hybrid males and the seven “possible hybrid” males showed haplogroup YA.

Morphology and mixed ancestry

Of the five adult intermediate mixed ancestry gibbons, three were female and two were male. As expected for traits under strong genetic control, these individuals showed mixed/mosaic characteristics in pelage (Fig 4A–4E) as well as vocalization patterns (S2 Fig). The five hybrid gibbons showed fluffy hair (Fig 4A–4E), which is more characteristic of white-handed gibbons (S3 Fig) than the straighter hair of pileated gibbons (S4 Fig). The pelage coloration of two of the females was similar to one another (Fig 4A and 4B) while it was different in the third female. Their main body hair was creamy buff, similar to buff white-handed gibbons, while their ventral hair was black, similar to adult pileated gibbon females. Their head and face ring pelage color patterns were similar to those of young adult pileated females who still express white eyebrows. The hair on the hands and feet was white, which is typical for both species, but the white hair extended up until the middle of the forearms, which is further than in typical white-handed gibbons, on which the white area usually reaches only around the wrists and ankles only, and more consistent with pileated gibbon’s extent of white hair on the forearm. At the same time, the black hair on the chest and abdomen also extended to the internal part of proximal upper limbs, which is absent in typical adult pileated gibbon females. The third female’s pelage pattern showed much more black hair on the entire body than the other two females, while her eyebrows, cap, hands and feet hairs resembled the white hair of the other two females (Fig 4C). The two adult hybrid males were similar to adult male pileated gibbons in that they expressed a white crown and white genital tuft, but were similar to white-handed gibbons in the expression of the white face ring and white hands and feet (Fig 4D and 4E).

Fig 4

External morphology of hybrid gibbons.

(A) Adult female hybrid of AA1. (B) Adult female hybrid of AB1. (C) Adult female hybrid of AC4. (D) Adult male hybrid of AA2. (E) Adult male hybrid of AA3. (F) Adult male hybrid of L. (G) Adult female hybrid of AC3. (H) Adult female hybrid of A. See Fig 3 for the degree of mixed ancestry.

Aligned with the SNVs analysis, other adult hybrid males and females with lower levels of mixed ancestry of pileated gibbon showed mosaic characteristics. For example, an adult male with 0.185 pileated ancestry showed a pelage color pattern of the black morph of white-handed gibbons but also showed a prominent white genital tuft not present in white-handed gibbons (Fig 4F). One adult female with 0.022 pileated ancestry also showed a color pattern similar to the black morph of white-handed gibbons, with a thinner white face ring (Fig 4G). One adult female with 0.088 pileated ancestry showed a typical buff white-handed gibbon pelage color (Fig 4H). Overall, three hybrid individuals with low levels of pileated ancestry were indistinguishable from typical white-handed gibbons, while another two hybrids with low levels of pileated ancestry showed atypical patterns. Among those, the extent of detected plieated ancestry and the presence/absence of atypical pelage color pattern were not related.

Pelage coloration of non-adult hybrid gibbons varied (S5 Fig). In addition to the six genetically analyzed, non-adult hybrid gibbons, there were several non-adult gibbons observed together with hybrid adults (Table 1). Many of them showed atypical characteristics for either species and thus appeared to be the offspring of hybrids. Some juveniles and adolescents showed a color pattern similar to pileated gibbons: whitish-grey with black patches on the abdominal area and the top of the head (S5 Fig).

Table 1

Gibbon groups with adult hybrid(s).

Group	Adult female	Adult male(s)	Offspring of the hybrid	Other non-adult members
AA1	Hybrid†	H. lar† (primary)	4 (3† and 1‡)
		“Possible hybrid”† (secondary)
AA2	H. lar‡	Hybrid†	2‡
AA3	H. lar‡	Hybrid† (primary)	1‡	H. lar† subadult female
		H. lar† (secondary)
AB1	Hybrid†	H. pileatus‡	1‡
AC3	Hybrid† (backcross with H. lar)	H. pileatus†	3 (2† and 1‡)
AC4	Hybrid†	“Possible hybrid”§	2 (1† and 1‡)
A	Hybrid§ (backcross with H. lar)	H. lar† (primary)	2¶ (both disperesd before 2010)
		H. lar† (secondary)
L†	H. lar	Hybrid† (backcross with H. lar) (primary)		H. lar¥ subsdult female
				H. la¥juvenile
				H. lar¥ infant
		H. lar† (secondary)
R (status in 2010)	Hybrid§ (backcross with H. lar)	H. lar†	1¶
W (status in 2010)	Hybrid§ (backcross with H. lar)	H. lar†	2¶

†: genetically and phenotypically confirmed

‡: phenotypically confirmed

§: mixed status was genetically confirmed, but not detected in phenotypes

¶: based on previous kinship analyses [16]

¥: genetically not tested.

Vocalization and mixed ancestry

As previously reported, both male and female intermediate hybrid gibbons sang phrases different from those of pure white-handed or pileated gibbons [15, 32, 54]. This was especially detectable in the female great call–male coda sequence (S4 Fig) but also other phrases. There were atypical notes among hybrid gibbons, including non-adult individuals with various levels of mixed ancestry.

Group composition of white-handed gibbons, pileated gibbons and their hybrids

Both, adult hybrid females and males formed groups (Table 1). The intermediate hybrid females formed a pair or a multi-male/one-female trio with varying male types (groups AA1, AB1 and AC4). Intermediate hybrid males formed a pair or a multi-male/one female trio with a white-handed female and a white-handed secondary male (groups AA2 and AA3). Although we found no intermediate hybrid male paired with a pileated female, this result was probably an outcome of our small sample size and white-handed gibbons mainly occupied the study area. As previously explained (i.e., see sections of external morphology), all five adult intermediate hybrid gibbons were assumed to have reproduced because many non-adult gibbons in their groups equally showed external morphologies inconsistent with either pureblood-species (S5 Fig).

The other five hybrid gibbons with lower levels of pileated ancestry also formed groups (Table 1). Four of them formed groups with white-handed gibbons, and the last formed a group with a pileated gibbon. The presence of putative offspring in several of the groups suggested that at least four of the five gibbons had reproduced.

We found five groups formed with two different species (Table 2), of which four included individuals who could not be genotyped because of missing fecal samples. However, pelage color and vocalization of non-genotyped groups suggested no or minimal mixed ancestry. Of these groups, only one was formed by a pair with one pileated female and one white-handed male (group AB3). Three were multi-male groups: each with a white-handed and a pileated gibbon male paired with a white-handed gibbon female (groups AB2, AB5 and AD1). The remaining group was composed of a white-handed and a pileated gibbon female with two white-handed gibbon males (group AA10). In those five groups, no hybrid offspring were detected (Table 2).

Table 2

Gibbon groups made up of heterospecific members.

Group	Adult female	Adult male(s)	Non-adult members
AA10	H. lar	H. lar (primary)	None
	H. pileatus	H. lar (secondary)
AB2	H. lar	H. lar (primary?)	H. lar juvenile
		H. pileatus (secondary?)
AB3	H. pileatus	H. lar	H. pileatus subadult female
AB5	H. lar	H. lar (primary?)	H. lar adolescent, H. lar juvenile
		H. pileatus (secondary?)
AD1	H. lar	H. lar (primary?)	H. lar subadult male (?), H. lar adolescent, H. lar juvenile
		H. pileatus (secondary?)

Discussion

Although, we previously reported the presence of phenotypical white-handed gibbons with a pileated-type mtDNA among Khao Yai gibbons [34], we were unable to address the levels of admixture because of a lack of autosomal DNA information at that time. In this study, we have identified hybrid gibbons with various levels of mixed ancestry by using autosomal SNV markers and mtDNA. Based on the estimated admixture levels, hybrid gibbons could represent a spectrum of first filial to plural backcross generations. Interestingly, four adult individuals (two males, two females) with white-handed typical phenotypic appearance but a pileated mtDNA haplotype (HKY11B) did not show mixed ancestries in their nuclear genomes, as represented by eight SNV markers (Fig 3). Thus, hybridization and introgression between white-handed and pileated gibbons in Khao Yai appears to be a rather non-recent event that has resulted from a series of repeated admixture events along multiple generations. The small number of SNV loci used in the present study together with the use of fecal samples restricted further investigations such as the number of generations passed after the initial hybridization event between the two species. Recent technological innovations in the use of fecal samples of wild primates for genome-wide analysis [55, 56] may help to obtain more information on the hybridization between the two gibbon species in Khao Yai.

We did not detect apparent survival or reproductive disadvantages in hybrid gibbons in the Khao Yai population and both male and female intermediate hybrids formed pair bonds with either white-handed or pileated gibbons. Furthermore, intermediate hybrid gibbons were with one to four non-adult individuals (mean = 2) in their groups with species-atypical phenotypes consistent with a genetically confirmed mixed ancestry for some individuals. The number of non-adult individuals (presumably offspring) observed in groups was within the range observed for white-handed gibbons (0–4) and pileated gibbons (1–3) in Khao Yai, Kaeng Krachan and Khao Soi Dao (the present study and [25]). These findings support the preliminary conclusion that the reproductive success of hybrids and parental species are not significantly different from one another. Moreover, the presence of subadult offspring suggests the stable position of the hybrids in the groups. However, only a molecular parentage analysis of paternity will be able to assess if hybrid males reproduce as effectively as males of parental species and likewise long-term molecular monitoring will be needed to assess reproducve rates in hybrid and parental species groups at Khao Yai.

Overall, in Khao Yai, a long history of hybridization and introgression between white-handed and pileated gibbons is strongly supported by our autosomal SNV analysis. A moderate tempo and mode of hybridization and introgression coupled with no apparent disadvantages in hybrids could have established the introgression pattern seen between the two species today. After the divergence of white-handed and pileated gibbons around 3–4 million years ago [35, 44, 57, 58], it is readily conceivable that hybridization and introgression occurred repeatedly where contact zones had formed along the species’ distribution boundaries. Together with behavioral data, genome-wide analyses focusing on Khao Yai gibbons, can shed further light on introgression and the evolutionary consequences of hybridization in small apes.

Median-joining network of hypervariable region 1 of mtDNA.

Short bars on branches indicate the number of substitutions between nodes. Node size reflects the number of each haplotype observed among 88 gibbons. HKY: 21 haplotypes observed at Khao Yai; HKK: 8 haplotypes observed at Kaneg Krachan; HKSD: 3 haplotypes observed at Khao Soi Dao.

(TIF)

Click here for additional data file.

Sonograms of female great call–male coda sequence of white-handed, pileated and hybrid gibbons.

(A) A white-handed gibbon pair at Kaeng Krachan. (B) A pileated gibbon pair at Khao Soi Dao. (C) A hybrid female and a white-handed male pair at Khao Yai. (D) A white-handed female and a hybrid male pair at Khao Yai.

(TIF)

Click here for additional data file.

Pelage color of white-handed gibbons.

(A) Adult male (black morphotype). (B) Adult female (buff morphotype) and infant (buff morphotype). (C) Adult female (black morphotype) and infant (black morphotype). (D) Juvenile (buff morphotype).

(TIF)

Click here for additional data file.

Pelage color of pileated gibbons.

(A) Adult male. (B) Adult female. (C) Adult female and infant. (D) Adolescent.

(TIF)

Click here for additional data file.

Pelage color of non-adult hybrid gibbons.

(A) Subadult male of AA1. (B) Adolescent female of AA1. (C) Juvenile female of AA1 (A–C: putative mother = intermediate hybrid). (D) Subadult male of AA2. (E) Juvenile of AA2 (D–E: putative father = intermediate hybrid). (F) Juvenile of AB1 (putative mother = intermediate hybrid). (G) Adolescent female of AC3. (H) Juvenile of AC3. (I) Infant of AC3 (G–I: putative mother = hybrid with a low level of mixed ancestry).

(TIF)

Click here for additional data file.

Status of SNV sites and primer–probe information.

(XLSX)

Click here for additional data file.

Samples and genotypes.

(XLSX)

Click here for additional data file.

9 Oct 2021

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Reviewer #1: Matsudaira and colleges present genetic and phenotypic evidence for hybridisation of white-handed and pileated gibbons in a hybrid zone.

Being unfamiliar with gibbon phenotypic diversity I restrict my comments to the genetic analysis which is within my area of expertise.

The authors present a genetic analysis, using 8 autosomal, MT and Y markers. This appears a novel investigation of a recent and/or ongoing hybridisation of these two species, and a counterpart to the investigation of older introgression described in Matsudaira and Ishida Heredity volume 127, pages312–322 (2021).

As such, I would be pleased if ultimately this paper was published in PLoS One. There is however a question as to how strong the interpretation of the genetic data is.

I certainly would agree with the authors that even a small number of suitably chosen loci can readily differentiate between two diverged lineages, and that these would also provide some information as to introgression/hybrid formation and/or a cline of ancestry. I disagree that there is enough resolution to readily differentiate between F1/F2 or various generations of repeat backcrosses. Given the small number of autosomal markers (less than the number of chromosomes), and the associated uncertainty in estimated ancestry components - even with only K = 2 - many different population processes could result in very similar arrays of ancestry that the authors uncover within the hybrid zone. Thus, I think it is fair to conclude that the authors uncover genetic evidence for recent hybridisation (Recent being relative to the related findings in Matsudaira and Ishida 2021). I do not think that one can say, as the authors do in the Abstract "Four phenotypically white-handed gibbons showed introgressed pileated type mtDNA, but the autosomal SNV admixture was not detected" and therefore ... "some genomic portions have introgressed into the gene pool of the counterpart species". In my opinion, this is too strong an interpretation of their results. I would be much happier if statements of this kind came with a suitable disclaimer. For example: "These gibbons were not only F1 hybrids, but also ones with plural backcross generations" (Lines 405:406). Much better could be, "Our data suggests that these gibbons could be a mixture of F1 hybrids, and perhaps ones with plural backcross generations, but the limited resolution enabled by small number of marker loci cannot rule out other scenarios acting on shorter timescales, such as a mixture of F1/F2 individuals". Of course how the authors want to address this point is up to them. Also, please note this is but one example, and my comment should be interpreted as having the authors rethink all statements on genetic ancestry that are definitive in tone such as Lines 428-429.

Minor points:

Data availability. I searched all three databases and could not find the listed accessions. Can the authors please confirm the availability of data.

Selection of autosomal SNVs: please expand on the relevant methods. How many DNA sequences from GenBank were initially selected?; how many independent fixed differences did you find? How many additional loci came from Perelman et al?; In essence - the reader should have some clarity about how you came to only 8 SNVs.

Lines 70-71: citation for disadvantages of hybridisation.

Reviewer #2: In this manuscript, “Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone”, the authors seek to uncover the degree of introgression and mating patterns between two endangered gibbon species, white-handed and pileated gibbons. They genotyped 88 individuals including 72 individuals from the hybrid zone, 12 purebred white-handed and 4 purebred pileated gibbons, with 8 autosomal SNPs. They also determined 632 bp-sequence on mtDNA and one 2bp-indel on Y chromosome for most of the individuals.

However, as I explained below, I don’t think it’s possible to measure the extent of introgression genetically with this data because of the small number of SNPs and the small number of the purebred samples. Rather, the importance of this study is in proposing potential markers to distinguish the two species and in accumulating genotype information of these endangered species. I recommend to reorganize this manuscript in that way.

1. The major concern is the small number of SNPs analyzed in this manuscript (8 SNPs) and the small number of the purebred individuals (12 white-handed and 4 pileated gibbons). It is hard to say that these SNPs are fixed differences between these species. The authors chose the 8 autosomal SNPs from the previous works of other groups; 5 SNPs from Chan et al. (2013) and 3 SNPs from Perelman et al. (2011). But the numbers of the animals used in the papers are also very small. In Chan et al., 11 white-handed and 4 pileated gibbons were used, and in Perelman et al., only one white-handed gibbon is used. It means, the genotypes of the 8 autosomal SNPs are available only for 13 or 23 white-handed gibbons and 4 or 8 pileated gibbons in total. It is likely that the 8SNPs are not fixed differences between the species but are segregating at high frequency within each species. In that case, it’s not possible to distinguish the two species with these SNPs.

2. In lines 269-270 and in Fig 3., the authors mentioned “the threshold of backcross and pure white-handed gibbons”. How was it determined? Also, how the criteria between F1 and backcross was determined (Fig3)? I guess it came from the identification of species base on vocalization and morphology. If so, it means that in this manuscript, the species identification is not based on genetic data but based simply on the vocalization and morphology. The aim of this manuscript is written as analyzing the samples genetically to evaluate the extent of introgression, but the actual analyses are done the other way round. I think the authors’ true interest is in finding a set of potential markers to distinguish species for future studies and to confirm hybrids which are identified from vocalization and morphology. I do understand the importance of finding the markers for endangered species, for which it’s difficult (/not possible) to get good amount of DNA samples. But the manuscript needs to be modified to make the aim clear.

3. The sample information is not clear enough. The authors collected 129 fecal samples in Khao Yai, 71 fecal samples in Kaeng Krachan and 9 fecal samples on Khao Soi Dao (lines179-181). They also genotyped DNA samples previously collected in Khao Yai (lines 193-194). Then suddenly declared “Overall, 93 DNA samples of 88 individuals were genotyped” without any explanations. Please provide the full sample list with sample ID, sampling location, sex, age category, species identified by vocalization and pelage coloration, and information from genetic data. If the data includes the samples which were used in previous studies, please explain it with the same sample IDs as in the previous works.

4. I think the authors should explain about the endangered status of the two species more, if that is the reason why the amount of genetic data is very limited.

5. The authors wrote “The electropherograms of HVR1 were visualized by MEGA6 " (line 247). Is it a mistake? I couldn’t find electropherograms in this manuscript.

**********

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15 Nov 2021

Dear Dr. Gojobori,

Thank you very much for evaluating the manuscript entitled “Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone”. We revised the manuscript following your comments and the two reviewers. The major point is that we toned down the conclusion on the level of admixture of each individual due to the limited number of SNV markers. Please find the details of revisions in the replies below.

In addition, submitting the agreements from copyright holders of the map information for Fig1 and Fig2 were requested. However, we recreated the maps by using only the data provided under the CC BY-SA license and describe the information in the figure legends appropriately. Therefore, we believe the agreements are not needed for these cases.

Comments from the editor

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both of the reviewers agree on that the limited number of genetic markers in this study cannot provide enough resolution to clearly distinguish F1s and backcrossed individuals or to evaluate the degree of gene flow. I have to say that the conclusions are not fully justified by the data in the current manuscript. However, I agree with the reviewer #1 on that this manuscript is publishable if the authors can revise the manuscript and tone down the conclusion. Therefore, my decision is “Major Revision”.

There was a disagreement between the reviewers regarding the limited number of available SNPs or genetic markers. Considering that obtaining larger sample size than the current data is difficult, I am more inclined to agree with the reviewer #1. But as reviewer #2 suggested, please explain the difficulty of obtaining samples and justify the reason why the only the limited sample size and the limited number of genetic markers were used.

Answer: Thank you very much for your kind understanding on the difficulties in obtaining large number of both samples and appropriate markers. We revised the manuscript and added the explanation of difficulties in the sampling from unhabituated gibbons (line 187-196) and finding markers (line 211-234). We also agree with you and reviewer #1 at the point of toning down the conclusion because of the difficulties in distinguishing first filial (F1) and backcrosses. Therefore, we avoided concluding the individuals as F1 by replacing the sentences.

There was some unclear points on how the genetic markers were selected and which individuals are included in this study. Please clarify these points on the revised manuscript because these information is essential to make the manuscript technically sound.

Answer: We revised and added detailed information about the finding of markers (line 211-234).

Also please address the other points raised by the reviewers than I mentioned below.

Answer: We revised accordingly. Please find replies below for the details.

Review Comments to the Author

Reviewer #1: Matsudaira and colleges present genetic and phenotypic evidence for hybridisation of white-handed and pileated gibbons in a hybrid zone.

Being unfamiliar with gibbon phenotypic diversity I restrict my comments to the genetic analysis which is within my area of expertise.

The authors present a genetic analysis, using 8 autosomal, MT and Y markers. This appears a novel investigation of a recent and/or ongoing hybridisation of these two species, and a counterpart to the investigation of older introgression described in Matsudaira and Ishida Heredity volume 127, pages312–322 (2021).

As such, I would be pleased if ultimately this paper was published in PLoS One. There is however a question as to how strong the interpretation of the genetic data is.

I certainly would agree with the authors that even a small number of suitably chosen loci can readily differentiate between two diverged lineages, and that these would also provide some information as to introgression/hybrid formation and/or a cline of ancestry. I disagree that there is enough resolution to readily differentiate between F1/F2 or various generations of repeat backcrosses. Given the small number of autosomal markers (less than the number of chromosomes), and the associated uncertainty in estimated ancestry components- even with only K = 2 - many different population processes could result in very similar arrays of ancestry that the authors uncover within the hybrid zone. Thus, I think it is fair to conclude that the authors uncover genetic evidence for recent hybridisation (Recent being relativeto the related findings in Matsudaira and Ishida 2021). I do not think that one can say, as the authors do in the Abstract "Four phenotypically white-handed gibbons showed introgressed pileated type mtDNA, but the autosomal SNV admixture was not detected" and therefore ... "some genomic portions have introgressed into the gene pool of the counterpart species". In my opinion, this is too strong an interpretation of their results. I would be much happier if statements of this kind came with a suitable disclaimer. For example: "These gibbons were not only F1 hybrids, but also ones with plural backcross generations" (Lines 405:406). Much better could be, "Our data suggests that these gibbons could be a mixture of F1 hybrids, and perhaps ones with plural backcross generations, but the limited resolution enabled by small number of marker loci cannot rule out other scenarios acting on shorter timescales, such as a mixture ofF1/F2 individuals". Of course how the authors want to address this point is up to them. Also, please note this is but one example, and my comment should be interpreted as having the authors rethink all statements on genetic ancestry that are definitive in tone such as Lines 428-429.

Answer: Thank you very much for your kind comments on improving the manuscript. We agree with your comments that the small number of SNV markers used in the study has limitations in determining the generation of hybrids. We revised the manuscript and avoid the use of the term, F1, for the individuals who showed intermediate admixture levels. We also toned down the conclusion following the comments.

Minor points:

Data availability. I searched all three databases and could not find the listed accessions. Can the authors please confirm the availability of data.

Answer: The sequences were registered and thus the accession numbers were assigned by DDBJ. Opening the sequences for the public is pending. We will open the data as soon as the manuscript is accepted.

Selection of autosomal SNVs: please expand on the relevant methods. How many DNA sequences from GenBank were initially selected?; how many independent fixed differences did you find? How many additional loci came from Perelman et al?; In essence – the reader should have some clarity about how you came to only 8 SNVs.

Answer: We followed the comments and added detailed explanations for the screening process of SNVs (line 211-234).

Lines 70-71: citation for disadvantages of hybridisation.

Answer: We added the citation and an example of disadvantage (line 67-70).

Reviewer #2: In this manuscript, “Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone”, the authors seek to uncover the degree of introgression and mating patterns between two endangered gibbon species, white-handed and pileated gibbons. They genotyped 88 individuals including 72 individuals from the hybrid zone, 12 purebred white-handed and 4 purebred pileated gibbons, with 8 autosomal SNPs. They also determined 632 bp-sequence on mtDNA and one 2bp-indel on Y chromosome for most of the individuals.

However, as I explained below, I don’t think it’s possible to measure the extent of introgression genetically with this data because of the small number of SNPs and the small number of the purebred samples. Rather, the importance of this study is in proposing potential markers to distinguish the two species and in accumulating genotype information of these endangered species. I recommend to reorganize this manuscript in that way.

Answer: Thank you very much for your kind comments on the manuscript. We agree that our previous conclusion was overstated against the small number of SNV markers used in the study and thus some ambiguity in the resolution of evaluating the admixture level remained. However, as we can see in the results, STRUCTURE analysis successfully distinguished pureblood white-handed gibbons and pileated gibbons without prior information of the species status in these samples (i.e. STRUCTURE did not know which samples were of white-handed gibbons and which samples were of pileated gibbons). Therefore, the finding of the presence of hybrid individuals with various levels of admixture level is robust. Following the comments from the editor, Dr. Gojobori, and reviewer #1, we toned down the conclusion. We believe our revisions made the manuscript scientifically feasible.

1. The major concern is the small number of SNPs analyzed in this manuscript (8 SNPs) and the small number of the purebred individuals (12 white-handed and 4 pileated gibbons). It is hard to say that these SNPs are fixed differences between these species. The authors chose the 8 autosomal SNPs from the previous works of other groups; 5 SNPs from Chan et al. (2013) and 3 SNPs from Perelman et al. (2011). But the numbers of the animals used in the papers are also very small. In Chan et al., 11 white-handed and 4 pileated gibbons were used, and in Perelman et al., only one white-handed gibbon is used. It means, the genotypes of the 8 autosomal SNPs are available only for 13 or 23 white-handed gibbons and 4 or 8 pileated gibbons in total. It is likely that the 8 SNPs are not fixed differences between the species but are segregating at high frequency within each species. In that case, it’s not possible to distinguish the two species with these SNPs.

Answer: Technically saying, genetic markers used in STRUCTURE analysis do not have to be fixed in K species/populations. STRUCTURE assumes the presence of hidden K clusters among the samples, where each cluster has a particular frequency of each SNV. In this process, STRUCTURE does not know which sample comes from which cluster a priori. Therefore, if the number of SNV markers and the number of pureblood white-handed gibbons and pureblood pileated gibbons were too small to distinguish the two species, the membership coefficient shown by STRUCTURE should have not represented the ancestry of the two species. Fortunately, as we can see in the results, 12 gibbons from Kaeng Krachan and 4 gibbons from Khao Soi Dao showed a quite small amount of mixed ancestry which can be considered the noise usually observed in STRUCTURE analysis, and thus (it is safe to say, that) the number of SNV markers and number of pureblood gibbons used in the study was enough for detecting hybrid gibbons with admixed ancestry.

On the other hand, we agree that the number of the SNV markers was not enough to clearly distinguish first filial, second filial and several backcross generations as suggested by the editor and reviewer #1, we revised this point and toned down the conclusion.

2. In lines 269-270 and in Fig 3., the authors mentioned “the threshold of backcross and pure white-handed gibbons”. How was it determined? Also, how the criteria between F1 and backcross was determined (Fig3)? I guess it came from the identification of species base on vocalization and morphology. If so, it means that in this manuscript, the species identification is not based on genetic data but based simply on the vocalization and morphology. The aim of this manuscript is written as analyzing the samples genetically to evaluate the extent of introgression, but the actual analyses are done the other way round. I think the authors’ true interest is in finding a set of potential markers to distinguish species for future studies and to confirm hybrids which are identified from vocalization and morphology. I do understand the importance of finding the markers for endangered species, for which it’s difficult (/not possible) to get good amount of DNA samples. But the manuscript needs to be modified to make the aim clear.

Answer: The threshold of backcross and pureblood white-handed gibbons was determined based on the observed admixed ancestry values in 12 pureblood white-handed gibbons from Kaeng Krachan and 4 pureblood pileated gibbons from Khao Soi Dao. Because of the characteristic of STRUCTURE analysis, a small amount of mixed ancestry could be detected in pureblood samples. Therefore, the maximum admixed ancestry value 0.017 observed in one Kaeng Krachan white-handed gibbon was defined as the threshold. We added an explanation on this point (line 279-287). The threshold between the F1 and backcross was determined arbitrary and thus was inappropriate. Therefore, we revised and removed the distinction between F1 and backcross, and considered all the individuals as mere hybrids.

3. The sample information is not clear enough. The authors collected 129 fecal samples in Khao Yai, 71 fecal samples in Kaeng Krachanand 9 fecal samples on Khao Soi Dao (lines179-181). They also genotyped DNA samples previously collected in Khao Yai (lines 193-194). Then suddenly declared “Overall, 93 DNA samples of 88 individuals were genotyped” without any explanations. Please provide the full sample list with sample ID, sampling location, sex, age category, species identified by vocalization and pelage coloration, and information from genetic data. If the data includes the samples

which were used in previous studies, please explain it with the same sample IDs as in the previous works.

Answer: We restructured the section and made the number of individuals used in the analysis clear (line 187-196). We also added the information of individuals, fecal samples, genotypes etc. as Supplementary Table 2 following the comments from reviewer #2. We hope the additional information will help to understand the details of the subjects.

4. I think the authors should explain about the endangered status of the two species more, if that is the reason why the amount of genetic data is very limited.

Answer: The limitation of sample number is derived from the difficulty in collecting fecal samples from unhabituated gibbons (it took many days to collect fecal samples from each individual). Also, the limitation of the genetic markers was mainly due to the financial issue for designing primers and probes rather than the available sequences from the database.

5. The authors wrote “The electropherograms of HVR1 were visualized by MEGA6 " (line 247). Is it a mistake? I couldn’t find electropherograms in this manuscript.

Answer: Electropherogram is raw data of electrophoresis by a Sanger sequencing and thus usually not informative to be shown in the manuscript without particular reason (e.g. to emphasize the appearance of heterozygous sites in the raw data). It is possible to show an example of one sequencing reaction, but we don’t think it is essential for this manuscript. To avoid misunderstanding what the electropherogram is we revised the sentence and add an explanation (line 265-266).

Submitted filename: Response_to_Reviewers_20211115.docx

Click here for additional data file.

20 Jan 2022

Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone

PLOS ONE

Dear Dr. Matsudaira,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I generally agree with the two reviewers and I think they made a reasonable suggestions. I ask the authors to address the comments raised by the reviewers before this manuscript is published.

Please submit your revised manuscript by Mar 06 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

10 Feb 2022

Dear Dr. Gojobori,

Thank you very much for evaluating the revised manuscript entitled “Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone.” We revised our manuscript following comments of reviewers #3 and #4. A major revision point has been that we now adopt the term “possible hybrids” to describe some more ambiguous results in our analyses. We now also mention the limitation of the use of the small number of SNV loci in our study. We hope this revision now resolves reviewers’ concern of potential overstatement of results and thus we toned down some of our findings. Please find our detailed revisions in the reply to reviewers’ comments below.

Reviewer #3: I agree with both previous reviewers that the limited amount of data presented here should not be over-interpreted. In particular, I agree with the statement 1 of reviewer #2, the previously described SNPs might not represent fixed differences with the small number of individuals, especially since ancient admixture events were likely part of their history.

First, the observation in the STRUCTURE analysis could be explained by genetic clines or simply segregating variation rather than recent hybridization. There is one thing I would recommend in terms of analysis, which is a STRUCTURE analysis with higher k (3, 4, 5) to see if the supposed signature of mixing is not just a component restricted to individuals from this area.

Answer:

We performed STRUCTURE analysis with K = 3, 4, and 5. The analyses result almost equally divided “white-handed-gibbon ancestry” into 2, 3, and 4 components in each individual from the three study sites with no detectable change in “pileated-gibbon ancestry.” We interpret this to indicate that the supposed signature of mixing is not just a component restricted to individuals from Khao Yai National Park. We added the new information and analyses with K = 3, 4, and 5 in Methods and Results sections (Lines 248-253 and lines 273-278, respectively).

Second, the threshold for assigning “hybrid ancestry” seems somewhat low and arbitrary given the small number of loci. Inspecting Fig. 3, there are 5-7 likely hybrids with substantial ancestry from both species (if one assumes these loci are really fixed), while I would be more doubtful about the others. The results on morphology and pelage are lending good support to the hypothesis on observed hybrids, especially in the case of those with clear mixed ancestries. I would like to know if the “five hybrid individuals indistinguishable from typical white-handed gibbons” had a lower assignment of admixed ancestry than the three with atypical patterns (L373).

Answer:

There was no clear relationship between the degree of mixed ancestry and the presence/absence of an atypical pelage color pattern among individuals with low levels of mixed ancestry. We added the explanation in Results (Line 379-383).

Third, the discordance of mtDNA nuclear DNA might be pointed out. There are a few individuals with a haplotype assigned to H. pileatus which, in this restricted dataset, have nuclear ancestry only assigned to H. lar. This shows the difficulty in determining ancestries with such few markers. Compare that to humans and Neanderthals, where ancient Neanderthal admixture in modern humans has not been determined by obtaining several mtDNA sequences, nor by retrieving a small amount of nuclear DNA (Green et al., Nature 2006), but only by shallow genome sequencing (Green et al., Science 2010). I agree that it is difficult to obtain information from wild-living primates for this kind of studies, but it is a problem that the confidence in observations must be low when using such a small number of individuals for the reference populations and a very small number of genetic markers. In that sense, I would also add “putative” to any statement about admixture, from the abstract on throughout the paper. I.e. the phrasing should be even more cautious when referring to the genetic results.

Answer:

We followed this and the comment of reviewer #4, and revised corresponding sections to read “putative” when our results seemed ambiguous. Because there was no ambiguity in mixed ancestry among intermediate hybrids, we did not use “putative” to describe ancestry of those individuals. For other gibbons with low levels of mixed ancestry, we decided to recategorized them into “hybrids with low levels of mixed ancestry” and “possible hybrids”. We cannot fully exclude that some arbitrariness for making the decision between those two categories may remain, but considering the morphology and vocalization, the hybrid origin of “hybrids with low levels of mixed ancestry” seems apparent to us thus justifying the use of this category. We believe our revisions resolved the overstatement on the admixture of gibbons in this population.

Finally, technological innovations may likely offer opportunities to obtain much more information from fecal samples of wild primates, as has been shown in chimpanzees with sequencing after capture (see Fontsere et al., Molecular Ecology 2021). Considering the very finegrained picture that were obtained in humans and other primate species after sequencing at least substantial parts of the genome, in comparison to single markers or mtDNA sequences, I suggest the authors add a sentence into the discussion about that. This may in the future confirm hypotheses on hybridization gained from the limited data, although of course this also depends on funding.

Answer:

Following the suggestions, we added the limitation of the use of a small number of SNV markers and the prospects of applying new technologies to fecal genomic analysis of the Khao Yai gibbon population (Line: 443-448); we also added suggested references #55 and #56.

Some typos:

L191: fecals > fecal

L212: sentence broken

L317/319: who *were* identified

L349: futher > further

L427: not > to

L435: result*ed*

Answer:

We corrected all typos.

Reviewer #4: This manuscript is about investigating gibbons genetically in the hybrid zones. They make new genetic markers for identifying whether the individual is a “pure-blood” or a “hybrid”. They also discuss on the phenotypic traits for the individuals which were detected as hybrids.

This is a revised manuscript and I was not one of the original reviewers. I will make comments based on the previous reviewers’ comments and the author’s responses.

Overall, the previous comments made by the original reviewers make sense to me and the authors responses are satisfactory. I agree with the previous comments on the limitation of genetic markers. The authors efforts to tone down the conclusion was necessary to make the manuscript more technically sounding.

I have one concern on detecting hybrid using the result of STRUCTURE (e.g. fig 3).

The authors set the criteria for being hybrid as showing contributions of the minor ancestry at a level of more than 0.017. I think it is OK to label the individuals showing “intermediate” level of mixed ancestry as recent hybrids. However, it might be inappropriate to label individuals with marginal value of ancestry (i.e. 0.018 or 0.019) as recent hybrid. This is because the number of markers used is quite limited and even truly “pure-blood” individual may show more than 0.017 of minor ancestry level under this condition. Then I suggest the author to label the individuals with marginal value of ancestry as “possible hybrids” or so.

Answer:

Please, see our comment in response to reviewer #3 above. In sum, we adopted reviewers’ criticism and now introduced “possible hybrids” for individuals with marginal mixed ancestry.

Submitted filename: Response_to_reviewers_20220210.docx

Click here for additional data file.

14 Feb 2022

Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone

PONE-D-21-19052R2

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23 Mar 2022

PONE-D-21-19052R2

Introgression and mating patterns between white-handed gibbons (Hylobates lar) and pileated gibbons (Hylobates pileatus) in a natural hybrid zone

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