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Research Note

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

[version 1; peer review: 2 approved]
PUBLISHED 30 Aug 2017
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Abstract

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.

Keywords

Isopoda, cryptic species, Sarasota, Crustacea, Ligia exotica

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 baudiniana1,2. The species has been reported from Brazil3, the Caribbean islands4, the Pacific coastlines of Central and South America57, Bermuda8, Bahamas4, and in southern Florida9,10 and the Everglades4; 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 description11 focuses on characters that are of limited taxonomic importance12, lacks illustrations, and does not provide an account of male reproductive structures now known to be useful in Ligia taxonomy1214. 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 species15,16, 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 Andersson5, Rouse9, Schultz4,8, and Schultz and Johnson10. 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 region14. 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.

e2bf1152-2359-4b71-8298-50eb79ca3afa_figure1.gif

Figure 1. Ligia baudiniana (I) and its characteristic appendix masculina (II AD).

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 Keys9,10 and the Everglades4, 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 Schultz4,8 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.

SpeciesLocalityLabel or
haplotype name
LatitudeLongitudeGenBank
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, MexicoC1_220°25'13.64"N86°50'42.26"WKF555855N/A
L. baudiniana Indian Key, FL,
U.S.A.
C3_224°53'23.70"N80°40'31.38"WKF555859N/A
L. baudiniana Summerland
Key, FL,
U.S.A.
C4_124°39'07.62"N81°26'09.48"WKF555860N/A
L. baudiniana Nassau, The
Bahamas
C5_125°04'47.22"N77°22'11.52"WKF555858N/A
L. baudiniana Jaws Beach,
The Bahamas
C6_125°01'05.05"N77°32'49.00"WKF555862N/A
L. baudiniana Habana, CubaC7_2N/AN/AKF555861N/A
L. baudiniana Long Bird
Bridge, Bermuda
C10_432°21'05.34"N64°42'35.16"WKF555856N/A
L. baudiniana Stonehole Bay,
Bermuda
C12_132°15'19.62"N64°48'49.68"WKF555857N/A
L. baudiniana Fort Sherman,
Panama
A1_109°21'51.36"N79°56'55.56"WKF555844N/A
L. baudiniana Portobelo (B),
Panama
A2_109°32'14.72"N79°40'26.30"WKF555843N/A
L. baudiniana Portobelo (C),
Panama
A3_109°32'54.24"N79°40'14.10"WKF555846N/A
L. baudiniana Portobelo (A),
Panama
A4_109°33'11.70"N79°39'35.58"WKF555845N/A
L. baudiniana Yaguanabo, CubaA7_2N/AN/AKF555849N/A
L. baudiniana Playa Ancon,
Cuba
A8_1N/AN/AKF555848N/A
L. baudiniana Boca Chica,
Dominican
Republic
A9_118°26'37.02"N69°36'37.98"WKF555847N/A
L. baudiniana Playa Bonita,
Costa Rica
B1_110°00'39.59"N83°03'46.87"WKF555850N/A
L. baudiniana Piuta, Costa RicaB2_110°00'20.70"N83°02'06.92"WKF555851N/A
L. baudiniana Santa Marta,
Colombia
B4_211°20'07.74"N73°58'31.26"WKF555852N/A
L. baudiniana Piscaderabaai,
Curacao
D1_112°07'25.38"N68°58'09.30"WKF555866N/A
L. baudiniana Spaans Lagoen,
Aruba
D2_112°27'45.18"N69°58'00.42"WKF555865N/A
L. baudiniana Donkey Beach,
Bonaire
D3_112°07'50.10"N68°17'04.44"WKF555867N/A
L. baudiniana East Coast, ArubaD4_112°32'44.58"N69°57'46.68"WKF555868N/A
L. baudiniana Fajardo, Puerto
Rico
D5_118°21'38.84"N65°37'28.51"WKF555869N/A
L. baudiniana Veracruz,
Panama
E1_108°53'28.30"N79°35'35.19"WKF555863N/A
L. baudiniana Caldera, Costa
Rica
E2_109°56'26.96"N84°44'02.93"WKF555864N/A
L. baudiniana Buenaventura, I.
Palma, Colombia
G1_1N/AN/AKF555871N/A
L. baudiniana Maguipi,
Colombia
G1_2N/AN/AKF555870N/A
L. baudiniana Buenaventura, I.
Palma, Colombia
G2_1N/AN/AKF555872N/A
L. exotica Multiple localities
in China
CH12N/AN/AJX414150N/A
L. exotica Multiple localities
in China
CH13N/AN/AJX414151N/A
L. exotica Multiple localities
in China
CH14N/AN/AJX414152N/A
L. exotica Multiple localities
in China
CH15N/AN/AJX414153N/A
L. exotica Multiple localities
in China
CH16N/AN/AJX414154N/A
L. exotica Multiple localities
in China
CH17N/AN/AJX414155N/A
L. exotica Multiple localities
in China
CH18N/AN/AJX414156N/A
L. exotica Multiple localities
in China
CH19N/AN/AJX414157N/A
L. exotica Multiple localities
in China
CH20N/AN/AJX414158N/A
L. exotica Multiple localities
in China
CH21N/AN/AJX414159N/A
L. exotica Multiple localities
in China
CH22N/AN/AJX414160N/A
L. exotica Fort Johnson,
Charleston,
South Carolina,
USA
Ligia exoticaN/AN/AGU270929N/A
L. exotica Veracruz HarborOut_CAR30_119°11'40.19"N96°07'24.41"WKF546664N/A
L. exotica Indian Fields
Creek, Virginia,
USA
SERCINVERT037037°16'04.80"N76°33'21.69"WKU906047N/A
e2bf1152-2359-4b71-8298-50eb79ca3afa_figure2.gif

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 Alignment18 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 v7.0.1819, 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 clades14.

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
0.50-18.014.4-18.915.4-19.021.3-24.917.5-19.321.1-23.620.3-25.2
North
Caribbean
(C)
5.50 0.30-19.1
8.20
13.6-18.913.8-19.318.7-25.617.2-21.720.7-23.520.7-27.0
Central
American +
Antillean
(A)
16.216.0 0.30-7.80
4.20
13.8-16.220.0-23.720.0-23.717.8-21.623.5-27.1
South
American
(B)
17.417.215.0 0.30-5.20
3.50
20.2-25.219.6-22.822.2-23.923.7-30.0
Leeward
Antilles
(D)
23.122.621.421.7 0.80-17.0
12.8
19.1-23.621.0-23.922.4-27.8
Central
American
Pacific
(E)
18.518.921.721.221.0 N/A-N/A
5.20
21.1-23.221.8-27.9
Eastern
Pacific
(G)
22.322.219.722.822.722.3 0.30-1.00
0.70
22.5-24.1
L. exotica 23.423.725.225.025.225.523.2 0.00-14.9
5.10
e2bf1152-2359-4b71-8298-50eb79ca3afa_figure3.gif

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 displacements21.

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.

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Santamaria CA, Bischoff III ET, Aye M et al. First record of the Ligia baudiniana species complex in the American Gulf of Mexico Coastline, as confirmed by morphological and molecular approaches [version 1; peer review: 2 approved]. F1000Research 2017, 6:1602 (https://doi.org/10.12688/f1000research.12459.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
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Reviewer Report 16 Nov 2017
Stefano Taiti, Institute of Ecosystem Studies (ISE), National Research Council (Italy), Sesto Fiorentino, Italy 
Approved
VIEWS 10
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 ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Taiti S. Reviewer Report For: First record of the Ligia baudiniana species complex in the American Gulf of Mexico Coastline, as confirmed by morphological and molecular approaches [version 1; peer review: 2 approved]. F1000Research 2017, 6:1602 (https://doi.org/10.5256/f1000research.13491.r25492)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 11 Oct 2017
Mary K. Wicksten, Texas A&M University, College Station, TX, USA 
Approved
VIEWS 12
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 ... Continue reading
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Wicksten MK. Reviewer Report For: First record of the Ligia baudiniana species complex in the American Gulf of Mexico Coastline, as confirmed by morphological and molecular approaches [version 1; peer review: 2 approved]. F1000Research 2017, 6:1602 (https://doi.org/10.5256/f1000research.13491.r26865)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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