First record of the Ligia baudiniana species complex in the American Gulf of Mexico Coastline, as confirmed by morphological and molecular approaches.

Ligia isopods exhibit a constrained morphology that makes identification difficult. In the Greater Caribbean, a convoluted taxonomic history has left the distributional limits of Ligia baudiniana unclear. To date, no confirmed records of this species exist from the American Gulf of Mexico. Herein, we report the presence of L. baudiniana in Sarasota-Manatee Florida, as confirmed by morphological and molecular approaches. This is the first record of this species in the region and a ~300Km extension of its range. Specimens were collected in mangroves, underscoring the importance of protecting these habitats.

Introduction

The isopod genus Ligia includes ~40 nominal species, most of which inhabit a narrow range in the upper rocky intertidal habitats. In the Greater Caribbean Region (i.e. the Caribbean and adjacent regions), a single endemic species is currently considered valid: Ligia baudiniana [1, 2]. The species has been reported from Brazil [3], the Caribbean islands [4], the Pacific coastlines of Central and South America [5– 7], Bermuda [8], Bahamas [4], and in southern Florida [9, 10] and the Everglades [4]; however, doubt over historical records have left the distributional limits of L. baudiniana unclear.

L. baudiniana was described from specimens collected in the San Juan de Ulua Fort in Veracruz, Mexico. Milne-Edwards’ original species description [11] focuses on characters that are of limited taxonomic importance [12], lacks illustrations, and does not provide an account of male reproductive structures now known to be useful in Ligia taxonomy [12– 14]. Indeed, the terse description and source origin of the type material (i.e., artificial substrate) have led to confusion on whether L. baudiniana is a synonym of L. exotica or a valid species [15, 16], and to records and specimens identified as L. baudiniana to be re-classified as L. exotica ( 3 and references). This is particularly true for specimens from the American Gulf of Mexico coastlines, as most records appear to have been reclassified as L. exotica. Furthermore, a wide-ranging survey of Ligia in the Gulf of Mexico from Texas to Florida has shown artificial habitats in the region to harbor only L. exotica (unpublished study; Hurtado LA, Mateos M, Wang C, Santamaria CA, Jung J, Khalaji-Pirbalouty V, and Kim W).

The taxonomic confusion between L. baudiniana and L. exotica is complicated by the presence of a Ligia species endemic to habitats throughout the Greater Caribbean, Gulf of Mexico excluded, that is easily recognized by a unique male gonopod morphology that is readily distinguishable from L. exotica ( Figure 1), and that has been attributed to L. baudiniana by Andersson [5], Rouse [9], Schultz [4, 8], and Schultz and Johnson [10]. A recent molecular study demonstrated that Ligia exhibiting this trait form a well-supported monophyletic clade composed of several cryptic and highly divergent lineages endemic to the region [14]. The combination of these studies suggests that L. baudiniana as currently recognized: (a) is an endemic species to the Greater Caribbean Region; (b) can be identified using both molecular and morphological tools; and (c) appears to have a broad geographic range that includes the Caribbean islands, the Pacific coastlines of Central America to Ecuador, Bermuda, Bahamas, and southern Florida.

Figure 1.

Ligia baudiniana (I) and its characteristic appendix masculina (II A– D).

This trait was used to putatively identify specimens collected in this study. The appendix masculine of L. exotica is shown in II-E. Photographs in panel II are reproduced under a Creative Commons license from Santamaria et al. (2014) [14].

In southern Florida, L. baudiniana is reported from the Florida Keys [9, 10] and the Everglades [4], while no confirmed records from the American Gulf of Mexico exist to date. In this study, we use molecular and morphological approaches to identify specimens collected from Sarasota and Manatee counties in Florida as L. baudiniana. Our findings extend the confirmed range of this species ~300-km into the Gulf of Mexico coastline of Florida and represent the first confirmed record of L. baudiniana in the American Gulf of Mexico coastline.

Methods

Ligia specimens were collected by hand across the Sarasota-Manatee counties in Florida ( Table 1, Figure 2) and field preserved in 70% EtOH. No permits were necessary for collections. Specimens were identified to species by inspecting the morphology of the apex of the endopod of the second pleopod of 15–25 male Ligia specimens per site, with individuals putatively identified as L. baudiniana if they exhibited a large process bifurcating close to the apex of the appendix masculina ( Figure 1A), as proposed by Schultz [4, 8] and confirmed by Santamaria et al. [14]. A subset of specimens was deposited in the Invertebrate Collections of the Biodiversity Research and Teaching Collections (BRTC) at Texas A&M University ( http://brtc.tamu.edu/).

Table 1.

Localities included in analyses and corresponding species ID, geographic information, GenBank accession numbers, and BRTC voucher numbers when applicable.

New records are in bold.

Species	Locality	Label or haplotype name	Latitude	Longitude	GenBank Accession No.	Museum Voucher
L. baudiniana	End of Tiara Drive, Bradenton, FL, U.S.A.	SRQ1	27°24'45.48"N	82°34'56.60"W	MF668214 MF668218	TCWC 2-4741
L. baudiniana	Quick Point, Longboat Key, FL, U.S.A.	SRQ2	27°20'19.10"N	82°34'56.49"W	MF668216 MF668219 MF668220 MF668223	TCWC 2-4737
L. baudiniana	Joan M Durante Community Park, Longboat Key, FL, U.S.A.	SRQ3	27°24'56.40"N	82°39'19.65"W	MF668217 MF668222 MF668225	TCWC 2-4740
L. baudiniana	Leffis Key, Bradenton Beach, FL, U.S.A.	SRQ4	27°27'08.64"N	82°41'17.25"W	MF668224	TCWC 2-4739
L. baudiniana	Gulf Drive South, Bradenton Beach, FL, U.S.A.	SRQ5	27°27'21.07"N	82°41'36.37"W	MF668215 MF668221 MF668224 MF668226	TCWC 2-4738
L. baudiniana	Cozumel, Mexico	C1_2	20°25'13.64"N	86°50'42.26"W	KF555855	N/A
L. baudiniana	Indian Key, FL, U.S.A.	C3_2	24°53'23.70"N	80°40'31.38"W	KF555859	N/A
L. baudiniana	Summerland Key, FL, U.S.A.	C4_1	24°39'07.62"N	81°26'09.48"W	KF555860	N/A
L. baudiniana	Nassau, The Bahamas	C5_1	25°04'47.22"N	77°22'11.52"W	KF555858	N/A
L. baudiniana	Jaws Beach, The Bahamas	C6_1	25°01'05.05"N	77°32'49.00"W	KF555862	N/A
L. baudiniana	Habana, Cuba	C7_2	N/A	N/A	KF555861	N/A
L. baudiniana	Long Bird Bridge, Bermuda	C10_4	32°21'05.34"N	64°42'35.16"W	KF555856	N/A
L. baudiniana	Stonehole Bay, Bermuda	C12_1	32°15'19.62"N	64°48'49.68"W	KF555857	N/A
L. baudiniana	Fort Sherman, Panama	A1_1	09°21'51.36"N	79°56'55.56"W	KF555844	N/A
L. baudiniana	Portobelo (B), Panama	A2_1	09°32'14.72"N	79°40'26.30"W	KF555843	N/A
L. baudiniana	Portobelo (C), Panama	A3_1	09°32'54.24"N	79°40'14.10"W	KF555846	N/A
L. baudiniana	Portobelo (A), Panama	A4_1	09°33'11.70"N	79°39'35.58"W	KF555845	N/A
L. baudiniana	Yaguanabo, Cuba	A7_2	N/A	N/A	KF555849	N/A
L. baudiniana	Playa Ancon, Cuba	A8_1	N/A	N/A	KF555848	N/A
L. baudiniana	Boca Chica, Dominican Republic	A9_1	18°26'37.02"N	69°36'37.98"W	KF555847	N/A
L. baudiniana	Playa Bonita, Costa Rica	B1_1	10°00'39.59"N	83°03'46.87"W	KF555850	N/A
L. baudiniana	Piuta, Costa Rica	B2_1	10°00'20.70"N	83°02'06.92"W	KF555851	N/A
L. baudiniana	Santa Marta, Colombia	B4_2	11°20'07.74"N	73°58'31.26"W	KF555852	N/A
L. baudiniana	Piscaderabaai, Curacao	D1_1	12°07'25.38"N	68°58'09.30"W	KF555866	N/A
L. baudiniana	Spaans Lagoen, Aruba	D2_1	12°27'45.18"N	69°58'00.42"W	KF555865	N/A
L. baudiniana	Donkey Beach, Bonaire	D3_1	12°07'50.10"N	68°17'04.44"W	KF555867	N/A
L. baudiniana	East Coast, Aruba	D4_1	12°32'44.58"N	69°57'46.68"W	KF555868	N/A
L. baudiniana	Fajardo, Puerto Rico	D5_1	18°21'38.84"N	65°37'28.51"W	KF555869	N/A
L. baudiniana	Veracruz, Panama	E1_1	08°53'28.30"N	79°35'35.19"W	KF555863	N/A
L. baudiniana	Caldera, Costa Rica	E2_1	09°56'26.96"N	84°44'02.93"W	KF555864	N/A
L. baudiniana	Buenaventura, I. Palma, Colombia	G1_1	N/A	N/A	KF555871	N/A
L. baudiniana	Maguipi, Colombia	G1_2	N/A	N/A	KF555870	N/A
L. baudiniana	Buenaventura, I. Palma, Colombia	G2_1	N/A	N/A	KF555872	N/A
L. exotica	Multiple localities in China	CH12	N/A	N/A	JX414150	N/A
L. exotica	Multiple localities in China	CH13	N/A	N/A	JX414151	N/A
L. exotica	Multiple localities in China	CH14	N/A	N/A	JX414152	N/A
L. exotica	Multiple localities in China	CH15	N/A	N/A	JX414153	N/A
L. exotica	Multiple localities in China	CH16	N/A	N/A	JX414154	N/A
L. exotica	Multiple localities in China	CH17	N/A	N/A	JX414155	N/A
L. exotica	Multiple localities in China	CH18	N/A	N/A	JX414156	N/A
L. exotica	Multiple localities in China	CH19	N/A	N/A	JX414157	N/A
L. exotica	Multiple localities in China	CH20	N/A	N/A	JX414158	N/A
L. exotica	Multiple localities in China	CH21	N/A	N/A	JX414159	N/A
L. exotica	Multiple localities in China	CH22	N/A	N/A	JX414160	N/A
L. exotica	Fort Johnson, Charleston, South Carolina, USA	Ligia exotica	N/A	N/A	GU270929	N/A
L. exotica	Veracruz Harbor	Out_CAR30_1	19°11'40.19"N	96°07'24.41"W	KF546664	N/A
L. exotica	Indian Fields Creek, Virginia, USA	SERCINVERT0370	37°16'04.80"N	76°33'21.69"W	KU906047	N/A

Figure 2.

Locations sampled in Sarasota and Manatee counties, Florida.

Locations are: (SRQ1) End of Tiara Drive; (SRQ2) Quick Point; (SRQ3) Joan M. Durante Community Park; (SRQ4) Leffis Key; (SRQ5) Near Coquina Beach. Detailed locality information can be found in Table 1. The smaller panel presents the distribution of L. baudiniana lineages reported to date throughout the Caribbean and its adjacent region.

Morphological identifications were corroborated using a mitochondrial barcoding approach. We extracted total genomic DNA from pleopods/pereopods for a subset of individuals putatively identified as L. baudiniana using the ZR Quick-gDNA Miniprep Kit. Previously described primers and conditions were used to PCR-amplify and sequence a 658-bp fragment of the Cytochrome Oxidase I gene (COI, primers LCO1490/HCO2198; 17). Positive amplicons were cleaned and sequenced at the University of Arizona Genetics Core (UAGC). Sequences were assembled in Geneious R v8.1.7.

We combined nucleotide sequences produced in this study with publicly available ones for L. baudiniana and L. exotica ( Table 1). We used default settings to align the resulting dataset using the MUSCLE Alignment [18] tool in Geneious R v8.1.7. No signs of misaligned regions or pseudo-genes were observed in the resulting alignment. The final alignment was imported into MEGA v7.0.18 [19], where we estimated a neighbor-joining tree under Kimura’s 2-parameter model (hereafter K2P; 20) and uniform rates. Support for the relationships within the tree were estimated by conducting 1,000 bootstrap replicates. Lastly, we calculated K2P genetic distances between haplotypes produced by this study, L. exotica, and previously reported L. baudiniana clades [14].

Results

Molecular identifications produced results congruent with morphological identifications. We obtained 12 unique COI haplotypes from a total of 25 individuals putatively identified as L. baudiniana. Haplotypes produced in this study were highly similar to each other (COI K2P 0.00–2.81%, Table 2) and to those reported from localities in the Florida Keys, The Bahamas, northern Cuba, Cozumel, and Bermuda (COI K2P 0.50–6.08%, Table 2). Haplotypes were moderately to highly divergent from L. baudiniana from other localities in the Caribbean (COI K2P 14.44–24.90%, Table 2), and highly divergent from L. exotica (COI K2P 20.32–25.18%). The neighbor-joining analysis produced similar results ( Figure 3), nesting all haplotypes produced in this study in a well-supported clade (Bootstrap Support = 100) with the Clade C reported by Santamaria et al. [14]. All unique haplotypes have been deposited in GenBank ( Table 1).

Table 2.

Divergence estimates between and within Ligia baudiniana lineages as estimated by K2P distances.

The top diagonals show minimum and maximum divergences between lineages, with lower diagonals presenting average genetic distances between clades. Within-group divergences are shown in the middle diagonal (in bold) in the order: minimum, maximum, and average divergence.

	Sarasota- Manatee (SRQ)	North Caribbean (C)	Central American + Antillean (A)	South American (B)	Leeward Antilles (D)	Central American Pacific (E)	Eastern Pacific (G)	L. exotica
Sarasota- Manatee (SRQ)	0.00-2.80 1.20	0.50-18.0	14.4-18.9	15.4-19.0	21.3-24.9	17.5-19.3	21.1-23.6	20.3-25.2
North Caribbean (C)	5.50	0.30-19.1 8.20	13.6-18.9	13.8-19.3	18.7-25.6	17.2-21.7	20.7-23.5	20.7-27.0
Central American + Antillean (A)	16.2	16.0	0.30-7.80 4.20	13.8-16.2	20.0-23.7	20.0-23.7	17.8-21.6	23.5-27.1
South American (B)	17.4	17.2	15.0	0.30-5.20 3.50	20.2-25.2	19.6-22.8	22.2-23.9	23.7-30.0
Leeward Antilles (D)	23.1	22.6	21.4	21.7	0.80-17.0 12.8	19.1-23.6	21.0-23.9	22.4-27.8
Central American Pacific (E)	18.5	18.9	21.7	21.2	21.0	N/A-N/A 5.20	21.1-23.2	21.8-27.9
Eastern Pacific (G)	22.3	22.2	19.7	22.8	22.7	22.3	0.30-1.00 0.70	22.5-24.1
L. exotica	23.4	23.7	25.2	25.0	25.2	25.5	23.2	0.00-14.9 5.10

Figure 3.

Neighbor-Joining phylogram of COI haplotypes for Ligia baudiniana and L. exotica.

Molecular identifications of putative L. baudiniana samples from Sarasota were made using K2P distances. All haplotypes for Ligia from Sarasota-Manatee counties (denoted by an *) are placed with previously reported haplotypes from the North Caribbean Clade reported by Santamaria et al. [14] in a well-supported clade (values near nodes represent bootstrap support values). Branches are drawn to scale, with colors and labels corresponding with those used by Santamaria et al. [14]. The COI haplotype obtained from topotypes of L. baudiniana by Santamaria et al. [14] is denoted by a †.

Discussion

Morphological and molecular evidence confirm that our sampled individuals represent L. baudiniana. These new records represent the first confirmed presence of this species in the Gulf of Mexico coastlines of the USA and extend the recognized range of the species ~300 km northward from a previous confirmed record from Florida Bay. Positive identifications in this study were made using both morphological and molecular characters. These findings are important as Florida’s rich coastal biodiversity faces serious threats such as sea-level rise, introduction of alien species, urbanization, habitat loss, and species displacements [21].

All L. baudiniana specimens collected in our surveys were found in coastal mangrove forests with no specimens found in >10 surveyed artificial habitats. This suggests that coastal development in the American Gulf of Mexico may have led to the replacement of a native species with an introduced one via the removal of mangrove habitats for the establishment of artificial substrates. Additional work is needed to establish whether L. baudiniana is present in other mangrove habitats along the Gulf of Mexico, thus clarifying the northern limits of this species’ range.

This is a very interesting paper  which can provide clear differences between two species of Ligia which have been mixed up in papers published up to the middle of last century.

I agree that the main differential character between L. baudiniana and L. exotica is the shape of male pleopod 2 endopod, even if several other morphological characters differ between the two species (e.g. shape of telson). In the text and Figure 1 the male pleopod 1 endopod is called “appendix masculina” or “gonopod”. I would avoid using these terms, since they are not in use in the taxonomy of terrestrial isopods, also because they might not refer to the same appendage. In Ligia the male modifications are present mainly in the pleopod 2 endopod, but in other genera of Oniscidea they are present on the pleopod 1 endopod or even on both pleopod 1 and 2 endopods.

Ligia baudiniana is reported also from the Galapagos.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Very nice work, well supported with clear diagrams and photographs. Double-check spelling: Introduction line 4: doubt has or doubts have...I wonder if further work will show that the reports from the eastern Pacific constitute a sibling species but this information is not pertinent to acceptance of the current manuscript. It might be worthwhile to point out that mangroves either do not occur or do not support characteristic communities in the northern Gulf of Mexico.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.