Species limits and phylogeography of Newportia (Scolopendromorpha) and implications for widespread morphospecies.

The genus Newportia Gervais, 1847, includes some 60 nominal species distributed in the Caribbean islands and from Mexico to central South America. Modern keys to species and subspecies are available, greatly facilitating identification, but some species are based on few specimens and have incomplete documentation of taxonomically-informative characters. In order to explore genetic variability and evolutionary relationships within geographically-widespread morphospecies, specimens of Newportia (Newportia) stolli (Pocock, 1896) and Newportia (Newportia) divergens Chamberlin, 1922, two nominal species distinguished principally by differences in suture patterns on T1, were sequenced for mitochondrial 16S rRNA and cytochrome c oxidase subunit I (COI) genes from populations in southern Mexico, Guatemala, Honduras and Brazil. Newportia (Newportia) stolli is paraphyletic with respect to Newportia (Newportia) divergens within a clade from Guatemala, Honduras, and Chiapas (Mexico), most trees being consistent with a single loss of a connection between the anterior transverse suture on T1, whereas specimens of "Newportia (Newportia) stolli" from Brazil are not closely allied to those from the Mesomerican type area. The widespread morphospecies Newportia (Newportia) monticola Pocock, 1890, was sequenced for the same loci from populations in Costa Rica, Colombia and Brazil, finding that specimens from these areas do not unite as a monophyletic group. Samples of Newportia (Newportia) oreina Chamberlin, 1915, from different regions of Mexico form geographic clusters that resolve as each other's closest relatives. These results suggest that some widespread species of Newportia may be taxa of convenience more so than natural groupings. In several cases geographic proximity fits the phylogeny better than taxonomy, suggesting that non-monophyletic species do not result from use of inappropriate molecular markers. Molecular identification is possible for specimens missing taxonomically informative morphological characters, notably damaged specimens that lack the ultimate leg pair, a protocol that may also apply to other taxonomically difficult genera that are prone to damage (such as Cryptops).

Introduction

Gervais, 1847 is a species-rich Neotropical genus that belongs to the family , encompassing blind with 23 leg-bearing segments, pectinate second maxillary claws, and kinked and pineapple-shaped processes in the gizzard (Shelley and Mercurio 2005; Koch et al. 2009, 2010). has until recently been classified as one of two genera in the subfamily , distinguished from Chamberlin, 1915, by different forcipular structures (Chagas-Júnior 2011). Phylogenetic analyses based on multi-locus molecular sequence data have, however, indicated that nests within rather than being the sister group, as does another clade that had been assigned to a separate subfamily, the Mesoamerican (Vahtera et al. 2013).

The geographic distribution of (including , Crabill, 1977, and Shelley & Mercurio, 2005 as subgenera: Vahtera et al. 2013) extends from northern Mexico throughout Central America and the Caribbean islands to Paraguay. Most species of have tarsus 2 of the ultimate leg divided into five to nearly 40 tarsomeres, or with indistinct separation of tarsi 1 and 2. Currently some 60 nominal species or subspecies are recognised (Minelli et al. 2006 and onwards; Schileyko 2013). In many species, diagnostic features involve the spinose processes on the ultimate prefemora and femora and the number of tarsomeres, all inconvenient characters because individuals frequently lose these legs when collected.

We propose a solution to the taxonomic impediment of missing ultimate legs by using mitochondrial sequence data to supplement identifications. We also explore phylogeographic patterns within and between select species of from Mexico and Central America using parsimony and maximum likelihood methods. The resultant phylogenies allow the taxonomic value of purportedly diagnostic morphological characters to be evaluated and for the limits of morphospecies to be tested.

Methods

Thirty-four specimens of from Mexico, Guatemala, Honduras, and Costa Rica were sorted mostly from collections made by the LLAMA (Leaf Litter Survey of PageBreakMesoamerica) project, deposited in the Museum of Comparative Zoology (MCZ), Harvard University, Cambridge Massachusetts, USA and accessible through the dedicated data base MCZbase (http://mczbase.mcz.harvard.edu). All tissues were fixed in absolute ethanol and thus were amendable to DNA sequencing.

Identifications were made using the most recent key for (Schileyko, 2013), supplemented with taxonomic descriptions in modern literature (Schileyko and Minelli 1998; Chagas-Júnior and Shelley 2003), standard monographs (Attems 1930), original descriptions, and examination of type material designated by R. I. Pocock in The Natural History Museum (London) and or by R. V. Chamberlin in the MCZ.

LLAMA specimens keyed to either Pocock, 1890, (Pocock, 1896), Chamberlin, 1915, or Chamberlin, 1922. All LLAMA specimens were sequenced for two mitochondrial loci: 16S rRNA and cytochrome c oxidase subunit I (COI). These loci were selected because they vary both within and between species, and even between individuals from geographically close populations. The 34 LLAMA samples were supplemented with and sequences from our previous work (Vahtera et al. 2013), nine new specimens from five localities in Amazonas and Roraima, Brazil, and novel sequences for an individual of Pocock, 1893, from Ecuador (see Table 1 for morphospecies determinations and locality data).

Table 1.

Specimens sequenced in this study and their GenBank accession numbers. Institutional abbreviation: MCZ, Museum of Comparative Zoology, Harvard University. Bold font indicates new sequence data.

Species	Voucher ID number	Lab code	Voucher	Country (State)	16S	COI	Lat. (degrees)	Long. (degrees)
Newportia adisi	130770	-	MCZ	Brazil (Amazonas)	KF676465	KF676506	2.93355S	59.96611W
Newportia (Ectonocryptoides) quadrimeropus	130826	-	MCZ	Mexico (Jalisco)	HQ402494	HQ402546	-	-
Newportia collaris	18827	95a	MCZ	Brazil (Roraima)	KP099547	KP099504	0.99185N	62.15915W
Newportia divergens	98078	72	MCZ	Honduras	KP099524	KP099481	14.45748N	89.06819W
Newportia divergens	99129	75	MCZ	Honduras	KP099525	KP099482	14.45603N	89.06904W
Newportia divergens	98978	81	MCZ	Honduras	KP099526	KP099483	15.69449N	86.86339W
Newportia divergens	99154	82	MCZ	Honduras	KP099527	KP099484	14.48139N	87.53225W
Newportia divergens	88191	85	MCZ	Guatemala	KP099528	KP099485	14.5357724N	90.69427782W
Newportia divergens	89343	101	MCZ	Guatemala	KP099529	KP099486	14.94704N	89.27627W
Newportia divergens	89474	105	MCZ	Guatemala	KP099530	KP099487	14.53256659N	90.15252622W
Newportia ernsti ernsti	18828	94	MCZ	Brazil (Roraima)	KP099522	KP099479	1.01113S	62.11409W
Newportia ernsti ernsti	105917	-	MCZ	Dominican Republic	JX422692	JX422669	-	-
Newportia longitarsis stechowi	130774	LP2871	AMNH	French Guiana	JX422693	JX422670	4.506277N	52.058305W
Newportia monticola	130778	-	MCZ	Colombia	JX422694	JX422671	5.7095080242N	73.4601469617W
Newportia monticola	80065	40	MCZ	Costa Rica	KP099531	KP099488	8.40667N	83.32833W
Newportia monticola	80743	49	MCZ	Costa Rica	KP099532	KP099489	8.78658N	82.95987W
Newportia monticola	81355	55	MCZ	Costa Rica	KP099533	KP099490	8.94997N	82.83375W
Newportia monticola	21666	91	MCZ	Brazil (Roraima)	KP099534	KP099491	1.01113S	62.11409W
Newportia oreina	94265	57	MCZ	Mexico (Tamaulipas)	KP099535	KP099492	23.0344N	99.18697W
Newportia oreina	94726	58	MCZ	Mexico (Oaxaca)	KP099536	KP099493	17.89844N	96.36253W
Newportia oreina	94185	59	MCZ	Mexico (Tamaulipas)	KP099537	KP099494	23.0233N	99.2883W
Newportia oreina	93765	60	MCZ	Mexico (Tamaulipas)	KP099538	KP099495	23.0611N	99.21564W
Newportia oreina	93666	62	MCZ	Mexico (Tamaulipas)	KP099539	KP099496	23.00835N	99.28511W
Newportia oreina	95181	66	MCZ	Mexico (Oaxaca)	KP099541	KP099500	17.65934N	96.33426W
Newportia oreina	93981	68	MCZ	Mexico (Oaxaca)	KP099540	KP099497	17.89844N	96.36253W
Newportia pusilla	18758	86	MCZ	Ecuador	KP099542	KP099498	0.6083333S	77.8825W
Newportia pusilla	18824	90	MCZ	Brazil (Amazonas)	KP099543	KP099499	2.93349S	59.96895W
Newportia sp.	81282	54	MCZ	Costa Rica	KP099544	KP099501	8.94997N	82.83375W
Newportia sp.	18822	89b	MCZ	Brazil (Roraima)	KP099545	KP099502	0.99539S	62.15904W
Newportia sp.	18825	92	MCZ	Brazil (Roraima)	KP099546	KP099503	1.02897S	62.08722W
Newportia stolli	106516	37	MCZ	Guatemala	KP099510	KP099467	14.91852N	91.10458W
Newportia stolli	81361	42	MCZ	Guatemala	KP099511	KP099468	15.1144N	89.68046667W
Newportia stolli	81360	44	MCZ	Mexico (Chiapas)	KP099505	KP099462	16.13853333N	90.90146667W
Newportia stolli	79982	47	MCZ	Mexico (Chiapas)	KP099506	KP099463	16.96385N	91.59313W
Newportia stolli	80143	48	MCZ	Guatemala	KP099512	KP099469	15.0583333N	89.676667W
Newportia stolli	80175	50	MCZ	Mexico (Chiapas)	KP099507	KP099464	17.17536N	93.14939W
Newportia stolli	81363	52	MCZ	Mexico (Chiapas)	KP099508	KP099465	16.97416667N	91.58591667W
Newportia stolli	80208	53	MCZ	Mexico (Chiapas)	KP099509	KP099466	16.75181N	92.68267W
Newportia stolli	99225	71	MCZ	Guatemala	KP099514	KP099471	15.08405N	89.94991W
Newportia stolli	99279	78	MCZ	Guatemala	KP099513	KP099472	15.07708N	89.94795W
Newportia stolli	18826	88a	MCZ	Brazil (Roraima)	KP099520	KP099477	1.02897S	62.08722W
Newportia stolli	18830	93a	MCZ	Brazil (Roraima)	KP099521	KP099478	1.01113S	62.11409W
Newportia stolli	18827	95b	MCZ	Brazil (Roraima)	KP099523	KP099480	0.99185N	62.15915W
Newportia stolli	89566	99	MCZ	Guatemala	KP099515	KP099470	15.21241135N	90.21480799W
Newportia stolli	89321	100	MCZ	Guatemala	KP099516	KP099473	16.44568931N	89.54981728W
Newportia stolli	89306	102	MCZ	Guatemala	KP099517	KP099474	17.24033736N	89.62094017W
Newportia stolli	89355	103	MCZ	Guatemala	KP099518	KP099475	15.21318939N	90.21921316W
Newportia stolli	89606	104	MCZ	Guatemala	KP099519	KP099476	16.44147064N	89.53447W
Newportia stolli	130787	-	MCZ	Guatemala	KF676467	KF676508	15.0833333N	89.9441666W
Cryptops punicus	130604	-	MCZ	Italy	KF676461	KF676503	40.01471N	9.22261E
Scolopocryptops mexicanus	105626	-	MCZ	Ecuador	JX422703	JX422679	1.336111N	77.263055W

Total DNA was extracted from the legs utilizing the NucleoSpin®Tissue kit (Macherey-Nagel). Samples were incubated overnight. PCR amplifications were performed with illustra TM PuReTaq TM Ready-To-GoTM PCR Beads (GE Healthcare). The COI fragments were amplified using primer pair HCO1490 (Folmer et al. 1994) and HCOout (Carpenter and Wheeler 1999) and the 16S rRNA fragments using primer pair 16Sa/16Sb (Xiong and Kocher 1991; Edgecombe et al. 2002). The normal amplification cycle for COI consisted of an initial denaturation step (2 min at 95 °C), followed by 35 cycles of denaturation (1 min at 95 °C), annealing (1 min at 43 °C) and extension (1.5 min at 72 °C), followed by a final extension step (4 min at 72 °C). For the 16S rRNA fragment the cycle consisted of an initial denaturation step (2 min at 94 °C), followed by 35 cycles of denaturation (30 s at 94 °C), annealing (30 s min at 43 °C) and extension (1 min at 72 °C), followed by a final extension step (7 min at 72 °C). Visualization of the PCR products was done by 1 % agarose electrophoresis using Midori Green Advanced DNA Stain and FastGene® GelPic LED Box (Nippon Genetics, GmbH).

Samples were purified using ExoSAP-IT (Affymetrix) and sent to FIMM (Institute for Molecular Medicine Finland) for sequencing. Chromatograms were visualized and assembled using Sequencer 5.0.1 (Gene Codes Corp., Ann Arbor, Michigan, USA). Sequence alignment editor Se-Al (Rambaut 1996) was used to visualize the sequences simultaneously. GenBank registrations for new sequences are listed in Table 1.

Parsimony analysis was conducted with POY ver. 5.1.1 (Wheeler et al. 2014) run in 16 nodes in the high-performance supercluster Taito at CSC (IT-Center of Science), Finland. A timed search of three hours was first performed on the unaligned data set. PageBreakPageBreakPageBreakThe resulting tree was used as the starting tree for the next round in which an additional timed search of six hours was performed. Parameter set 111 (indel/transversion and transversion/transition costs all equal) was used throughout the searches and branch lengths were reported using the newly implemented command “report (“file_name.tre”, trees:(total, branches:true))”. Nodal support was calculated using parsimony jackknifing (Farris et al. 1996).

Additional analyses used a probabilistic approach with the maximum likelihood program RAxML ver. 8.0.22 (Stamatakis 2014). For these, multiple sequence alignments (MSA) were first estimated with MUSCLE ver. 3.6 (Edgar 2004) and then trimmed using Gblocks ver. 0.91b (Castresana 2000; Talavera and Castresana 2007) to remove areas of ambiguous alignment. Since COI sequences showed no length variation, they were not trimmed in Gblocks. The amount of 16S rRNA data that remained after trimming was 59% of the original 585 positions. The two data sets were concatenated using SequenceMatrix (Vaidya et al. 2011) and the concatenated data were analyzed with RAxML in the CIPRES Science Gateway (Miller et al. 2010). A unique general time reversible (GTR) model was specified for each partition independently. Nodal support was estimated using the rapid bootstrap algorithm (applying the Majority Rule Criterion) using the GTR-CAT model (Stamatakis et al. 2008).

Results

The combined analysis of both COI and 16S fragments using parsimony as the optimality criterion resulted in two most parsimonious (MP) trees of length 4625 steps. PageBreakThe strict consensus tree (Fig. 2) shows these two trees are almost identical, differing only in the placement of two Brazilian specimens of in relation to each other. Comparing strongly supported clades, the maximum likelihood tree (lnL -14054.372302: Fig. 3) shows the same major geographic and taxonomic groupings as the parsimony tree. This congruence is noteworthy because the data sets analyzed under these two optimality criteria were different (unaligned in POY and analyzed using the concept of dynamic versus static homologies with some regions removed in RAxML), as are the resampling methods (jackknifing and bootstrapping, respectively). Parts of the trees that are incongruent between the two analyses involve nodes that received low resampling supports in both analyses (e.g., the positions of and Brazilian specimen 89b relative to other species). Both analyses depict substantial branch lengths both within and between species, with only a few instances of no (or minimal) variation between specimens from the same or geographically close populations.

Figure 2.

Strict consensus of two optimal cladograms for under parameter set 111 for parsimony (POY) analysis. Abbreviations: BRA, Brazil; COL, Colombia; CR, Costa Rica; DR, Dominican Republic; ECU, Ecuador; FRG, French Guiana; GUA, Guatemala; HON, Honduras; MEX, Mexico.

Figure 3.

Maximum likelihood tree (lnL = -14054.372302). Abbreviations for countries as in Fig. 2.

Map of Mesoamerica, the Caribbean and northern South America showing geographic distribution of specimens analyzed herein (see Table 1 for coordinates of samples).

As in previous analyses based on sparser sampling for (Vahtera et al. 2013), () and () nest within in all PageBreakanalyses. Specifically, they unite with spp. that inhabit the same geographic region i.e., from the Brazilian Amazon groups within a clade composed of species of from there, whereas from Jalisco, Mexico, groups with the Mexican . These results reinforce proposals to classify , and presumably allied within and to regard as subordinate to (Vahtera et al. 2013). The traditional classification of and as separate genera because of their obvious phenotypic differences from N () might have predicted that they would be markedly different from genetically. However, neither nor depict long branch divergences from their closest relatives with respect to the studied loci, indeed being shorter than some population-level branches within species.

consists of two geographical clades and this division is found in both parsimony and likelihood analyses; one clade consists of all specimens from Tamaulipas (JK, BS 100) and the other of ones from Oaxaca (JK 100, BS 98). Interestingly, forms a well-supported (JK 99, BS 73) clade with the populations from Oaxaca, rendering PageBreakparaphyletic with respect to (and presumably ). A previous scolopendromorph phylogeny (Vahtera et al. 2013) had also indicated affinity between and ; analyses based on combined molecular and morphological data resolved them as sister-groups, although only one individual of each was then available. We note that possesses a shorter tarsus than most congeners. The phylogeny interprets the ancestral condition of the ultimate leg tarsi of as being elongate and divided into tarsomeres, with the relatively short tarsus 2 of being a possible precursor to the stout tarsi of the submerged taxon, “”. This transformation series increases the plausibility of the subclavate “ectonocryptopine” ultimate legs being derived from an ancestor with flagelliform tarsi, a result that was already strongly signaled by molecular phylogenies (Vahtera et al. 2013) and is reinforced by the current trees.

A Mesoamerican clade uniting and from Mexico (Chiapas), Guatemala and Honduras is recovered in both parsimony and likelihood analyses (Figs 2, 3), though resampling methods did not strongly support it (JK <50, BS 57). is resolved as monophyletic in the POY analyses but is nested within a paraphyletic , implying a single loss of the median part of the anterior transverse suture on T1 (Fig. 2). However, there is no jackknife support for the clade. In contrast, the likelihood analysis did not support monophyly of ; six individuals from Guatemala and Honduras resolve as a well-supported clade (BS 98), but two others from Honduras (81, 82) are grouped with two Mexican specimens, albeit with weak nodal support.

Specimens identified as from the Brazilian Amazon do not unite with supposed congeners from Mesoamerica but are instead most closely related to other taxa from the same region, i.e., a specimen identified as (91) and . This result implies that is polyphyletic and an indistinct segmentation of ultimate tarsus 2 has multiple (convergent) origins. This character had once served as the basis for recognising a subgenus , e.g., in the classification of Bücherl (1974), but this taxon is not used in current classifications (Schileyko and Minelli 1998). We re-examined the specimens again in light of the signal for non-monophyly in the phylogenetic analysis, attempting to recognize any morphological character(s) that would separate the specimens from Brazil from those from Mesoamerica. However, we found no distinctive characters between the samples; the specimens appear to be morphologically indistinguishable and using the existing keys they would all be identified as with confidence.

Costa Rican specimens of unite as a monophyletic group (JK 100, BS 99) in both analyses. In the maximum likelihood tree (Fig. 3) a Colombian specimen of (103974) is resolved as a sister taxon to the Costa Rican clade but this relationship is not found in the parsimony tree (Fig. 2). In neither analysis did a Brazilian specimen identified as unite with the other supposed conspecifics.

The two included specimens of , one from Ecuador (specimen 86) and the other from Brazilian Amazonas (specimen 90), likewise do not form a clade PageBreakbut instead are situated in different parts of the tree. The Brazilian specimen conforms to “Amazonian type ” of Schileyko and Minelli (1998), characterized by rudimentary paramedian sutures on T1 (in contrast to their complete absence in other populations). Both analyses group this Brazilian specimen together with but since there is no strong resampling support in either analysis (JK <50, BS 69), the question about its identity and closest relative remains unclear.

We also included a few specimens that could not be identified morphologically since they lacked ultimate legs, were juveniles, or did not key out to any known species. A specimen (54) from Costa Rica has a unique character combination and is apparently a distinct species but lacks its ultimate legs. In the POY analysis it groups together, although with weak support, with the Costa Rican clade. A very distinctive Brazilian specimen (89b) with all tarsi bipartite and tarsus 2 of the ultimate leg undivided groups at the base of the Mexican / clade in the parsimony analysis. However, there is poor resampling support for this grouping and it is instead allied to species with indistinctly segmented ultimate tarsus 2 and the Brazilian clade in the likelihood tree. The poor support values and topological instability under different analytical conditions render the affinities of this undescribed species uncertain.

Discussion

Some of the specimens used in this study were either of small size because of the collection methods employed (and thus may not have been appropriate for keying using traditional criteria formulated for mature specimens) or were missing their taxonomically-informative ultimate legs. Nonetheless, several such specimens could be identified with a high degree of accuracy because their sequence data placed them within clades whose nomenclature could be established based on standard external morphological characters. An example is provided by a juvenile from Brazil (92) that is in poor condition and cannot be identified to species. However, the analysis shows it to be a juvenile of a Brazilian clade assigned to . This approach is likely to be valuable in other groups of taxonomically-difficult centipedes that rely heavily on characters of the ultimate leg pair but often lack those legs in fixed specimens, such as , where the numbers of tibial and tarsal saw teeth are fundamental taxonomic characters. The identification of developmental stages or adults without key taxonomic characters is becoming standard for many groups of animals, including other arthropod groups, such as insects (Monaghan et al. 2009; Gattolliat and Monaghan 2010) and arachnids (Fernández et al. 2014).

Some morphologically delimited species were found to be monophyletic groups, like in the parsimony analysis, but others were paraphyletic or polyphyletic. This could be interpreted as a failure of the taxonomic characters traditionally used to delimit species or a failure in reconstructing an accurate tree by the markers selected. The second option is unlikely for the reasons outlined below, especially the biogeographical patterns exhibited in many clades where “distinct” species from the same regions tend PageBreakto cluster together and not with their supposed conspecifics from other geographical regions. In particular formed a series of geographic groupings that in part were paraphyletic with respect to sympatric species (specifically, to in Mesoamerica) or in other cases were found to be distantly related (Brazilian “”). The first pattern is consistent with being a grade united by a plesiomorphy (a continuous anterior transverse suture on T1), some parts of which are most closely related to a species defined by an apomorphic state (i.e., loss of the median extent of the anterior transverse suture). The tree topology, however, suggests that the Brazilian specimens identified as are misidentified. is likewise a questionable taxon, the monophyletic Costa Rican group never uniting with a specimen of the same putative species from Brazil and only variably so with one from Colombia. Brazilian and unite in a well-supported clade (JF and BS 100), indicating that, in this instance, geography is a better predictor of relationships than taxonomy. It is noteworthy that and are among the most geographically widespread “species” of , but our results suggest that the wide distribution is partly an artifact of morphologically-based identifications. The same evidently applies to , a morphospecies that is regarded as ranging from St. Vincent through Colombia to the Brazilian Amazon (Schileyko and Minelli 1998; Chagas-Júnior et al. 2014). Polyphyly of this species in the molecular trees suggests that its diagnostic characters (absent or rudimentary paramedian sutures on T1 and a lack of ventral spinose processes on the ultimate leg femora) evolved convergently in different regions.

Centipede systematics, still strongly influenced by mid 20th Century conceptualisations of species (see Edgecombe 2007), primarily assumes polymorphic and geographically widespread entities. The existing concepts that and are widespread throughout much of Central and South America exemplify where morphospecies do not appear to correspond to clades but rather are classes defined by combinations of characters. In these instances, molecular tools may prove to be invaluable for species delimitations, and novel morphological characters will need to be identified to rediagnose polyphyletic species.