A glimpse into a remarkable unknown diversity of oniscideans along the Caribbean coasts revealed on a tiny island

In this study, we report the results of a taxonomic survey of terrestrial isopods from Isla Grande, a ca 2 km2 island located in the north of Cartagena de Indias, Colombia. We found a total of 17 species belonging to nine families and 10 genera. Eight of these species have been reported only from the Caribbean region, nine are recorded for the fi rst time in Colombia, and three are new to science and described here: Tylos negroi López-Orozco, Carpio-Díaz & Campos-Filho sp. nov., Stenoniscus nestori López-Orozco, Taiti & Campos-Filho sp. nov. and Armadilloniscus luisi Carpio-Díaz, Taiti & Campos-Filho sp. nov. Our revision also determined that the genus Microphiloscia is a junior synonym of Halophiloscia; and moreover, Halophiloscia trichoniscoides comb. nov. is redescribed. We also provide illustrations for Armadilloniscus caraibicus and Armadilloniscus ninae. Most (16) of the species were found in coastal habitats (i.e., beaches, coastal lagoons and mangroves), whereas the tropical dry forest harbored only two species. Molecular phylogenetic inferences supported the presence of three species of Tylos in Isla Grande (i.e., one new species and a new lineage within each of two known species). Our work on Tylos highlights the importance of combining taxonomic and molecular analyses to support taxonomic decisions and uncover cryptic diversity. Due to the multiple threats to Caribbean coastal habitats, taxonomic and molecular genetic research are urgently needed to understand biodiversity patterns of oniscideans in the Caribbean, which will inform strategies for their protection. Such studies will also contribute to our knowledge of their evolution, ecology, and potential uses, as well as the factors that have shaped the remarkable Caribbean biodiversity.


Introduction
Terrestrial isopods (Oniscidea Latreille, 1802) constitute one of the most diverse groups within Isopoda Latreille, 1817, including more than 3800 species in 38 families and more than 500 genera (Schmalfuss 2003;Javidkar et al. 2015Javidkar et al. , 2017Sfenthourakis & Taiti 2015). They appear to form a monophyletic group, with the exception of the predominantly marine supralittoral genus Ligia Fabricius, 1798, which, according to recent molecular phylogenetic analyses, seems to be more closely related to marine isopods (Lins et al. 2017;Dimitriou et al. 2019). Pending a taxonomic revision, however, in this study we treat Ligia as being within the Oniscidea. Oniscideans are distributed in almost all terrestrial habitats, from the marine supralittoral to high mountain ranges, and from tropical forest to deserts, being absent in polar regions (Warburg 1993;Schmalfuss 2003;Sfenthourakis et al. 2020;WoRMS 2020a). A very large number of oniscideans remains to be discovered, but the taxonomic impediment is still a major cause of the delay in the recognition of this fauna (Ebach et al. 2011;Campos-Filho et al. 2014Coleman 2015). Recent molecular studies have uncovered high levels of cryptic diversity within nominal species of marine coastal oniscideans that were considered broad-ranging, but that instead encompass many genetically highly differentiated lineages, each with relatively small ranges (Hurtado et al. 2010Eberl et al. 2013;Santamaria et al. 2013Santamaria et al. , 2014. Their high potential for diversifi cation stresses the importance of continuing taxonomic and systematic studies of oniscideans in coastal habitats, to better recognize their patterns of diversity, which, in turn, will serve as the foundation for understanding aspects of their ecology, evolution and potential uses García-Hernández et al. 2015;Mattos et al. 2018). In addition, coastal habitats are under high pressure from sea level rise, pollution, and habitat destruction and modifi cation (e.g., Guarderas et al. 2008;Lorde et al. 2013). Thus, it is urgent to identify the diversity of coastal oniscideans to better design conservation strategies, which can in turn benefi t other species in this environment (Hurtado et al. 2010Santamaria et al. 2013).
The Caribbean region is considered a natural laboratory for research on biodiversity evolution. A complex geological history, along with other factors, has contributed to a remarkable diversity of marine and terrestrial organisms, the highest in the Atlantic Basin (Myers et al. 2000;Roberts et al. 2002;Brummitt & Lughadha 2003;Kerswell 2006;Ricklefs & Bermingham 2008;Miloslavich et al. 2010). Although oniscideans have long been considered a group that can be highly informative on Caribbean biogeography (Rosen 1975), they have been poorly studied in this region. Two recent molecular phylogenetic studies showed a high level of diversifi cation of coastal oniscideans in this region. Santamaria et al. (2014) identifi ed a phylogenetic clade of supralittoral Ligia distributed in the Caribbean Sea, Bahamas, southern Florida, Bermuda, and the Pacifi c coast of Central America and Colombia, which represented the range of Ligia baudiniana Milne-Edwards, 1840. The authors recovered seven highly divergent lineages within this clade, probably corresponding to different species, fi ve of which were observed in the Caribbean and two in the Pacifi c. Similarly, high levels of genetic differentiation have been reported for some Caribbean populations of another supralittoral oniscidean, Tylos sp., even over relatively short geographic distances . However, sampling of Tylos Audouin, 1826 was very limited and more efforts are needed throughout the Caribbean Sea to better understand the diversity of this taxon in the region. These two studies suggest that a high diversity of coastal oniscideans in the Caribbean is yet to be discovered, underscoring the importance of taxonomic and molecular systematics studies for these organisms in the region.
Despite an extensive coastline of about 1600 km of the Caribbean Sea in Colombia, studies of the diversity of oniscideans in coastal habitats have been very limited.  recorded Porcellionides pruinosus (Brandt, 1833) from San Andrés Island, a Colombian island located about 190 km off the coast of Nicaragua. López-Orozco et al. (2014) recorded L. baudiniana from Cartagena de Indias, northern Bolívar. Carpio-Díaz et al. (2016) recorded Tylos niveus Budde-Lund, 1885 and P. pruinosus from Barú Island, northern Bolívar. López-Orozco et al. (2017) described Pulmoniscus turbanaensis López-Orozco, Carpio-Díaz & Campos-Filho, 2017 from northern Bolívar, including specimens from Tierra Bomba Island. In addition, one of the fi ve Ligia clades identifi ed by Santamaria et al. (2014) in the Caribbean was observed on the coast of the Magdalena Department of Colombia.
Herein, we conducted a taxonomic survey of the oniscideans present at littoral, mangrove, and inland habitats of Isla Grande, a tiny island off the Caribbean coast of Colombia. Detailed external morphology was examined for all samples. In addition, we conducted molecular phylogenetic analyses of specimens of Tylos. Based on these examinations, three new species of oniscideans are described, and one previous junior synonym is redescribed. Full illustrations of the new and several species are provided as well as a taxonomic key for the identifi cation of all species. Our study highlights the immense potential of combined taxonomic and molecular systematics studies to uncover the diversity of oniscideans in coastal habitats of the Caribbean Basin, a region that needs urgent attention due to the multiple threats to its conservation.   Schultz, 1984. 9. Halophiloscia trichoniscoides (Vandel, 1973) comb. nov. 10. Littorophiloscia amphindica Taiti & Ferrara, 1986. 11. Littorophiloscia denticulata . 12. Littorophiloscia tropicalis . 13. Trichorhina heterophthalma Lemos de Castro, 1964. 14. Trichorhina bermudezae Carpio-Díaz, López-Orozco & Campos-Filho, 2018. Porcellionides pruinosus (Brandt, 1833). 16. Agnara madagascariensis (Budde-Lund, 1885). 17. Ctenorillo tuberosus (Budde-Lund, 1904).

Taxonomy
Two sampling methodologies were used. Direct Intuitive Searches (Taiti & Wynne 2015), implemented at all sites, consisted of hand-collection of specimens during searches in the sand and decomposing organic matter, roots and bark of trees, fallen logs and under rocks. The estimated time of the searches was about 20 minutes per observer (three observers) at each sampling site. In the TDF, we also used a Winkler sack (mesh size: 1 cm) to screen leaf litter; extraction of specimens was performed immediately by spreading the material on a white cloth. The specimens were preserved in 75% ethanol. The identifi cations were based on morphological characters with the use of micropreparations. The illustrations were made from photographs taken with Axio Lab A1 microscope and SteREO Discovery.V12 ZEISS stereo microscope with an adapted Axiocam ERc 5s camera and the aid of a camera lucida mounted on Wild M5 and M20 microscopes. The fi nal illustrations were prepared using the software GIMP (ver. 2.8) with the method proposed by Montesanto (2015Montesanto ( , 2016. For the already described species, material examined and distributions are presented. The synonymy lists include original descriptions and publications mentioning species occurring in Colombia. Additional references containing relevant taxonomic and distribution data are also included when necessary. For each new species, the type material, etymology, description, remarks, and distribution are given. The material is deposited in the research laboratories of the Biology program at Cartagena University, Cartagena, Colombia (CUDC-CRU) and the Collection of Isopod Crustaceans of the Instituto de Ciencias Naturales, National University of Colombia, Bogotá, Colombia (ICN-CR-is). Specimens from Cuba are deposited in the Museo di Storia Naturale, Sezione di Zoologia "La Specola", Florence, Italy (MZUF).

Molecular phylogenetic analyses of specimens of Tylos Audouin, 1826
Genomic DNA was isolated from 1 leg for seven specimens of Tylos with the DNEasy kit, following the manufacturer's protocol (Qiagen Inc., Valencia, CA). These specimens represented the three different species identifi ed by taxonomic analyses and different localities where they were sampled (see Results): Tylos marcuzzii Giordani Soika, 1954 from La Punta; Tylos niveus from Laguna Caracolí and Caño Ratón; and Tylos negroi López-Orozco, Carpio-Díaz & Campos-Filho sp. nov. from Playa Libre, El Silencio and La Punta-El Terminal. We attempted to PCR-amplify fragments of the mitochondrial genes 16S rDNA (using primers 16Sar and 16Sbr) and 12S rDNA (using primers Crust-12f and Crust-12r; primers and PCR conditions in Hurtado et al. 2013). PCR-amplifi ed products were cleaned with Exonuclease and Shrimp Alkaline Phosphatase, and subsequently cycle sequenced using the Sanger method. We used the software Sequencher ver. 4.8 (Gene Codes, Ann Arbor, MI) for sequence editing and primer removal.
The sequences obtained were manually aligned with sequences of the 16S rDNA and 12S rDNA mitochondrial genes for 17 of the 21 nominal species of Tylos, reported in Hurtado et al. (2013Hurtado et al. ( , 2014. In addition, we included a 16S rDNA sequence from a specimen identifi ed as T. niveus collected at the type locality of this species in the Florida Keys, USA (24°53′24″ N, 80°40′33.6″ W). Based on preliminary phylogenetic analyses, we identifi ed the closest relatives to the new specimens to generate a smaller dataset (i.e., clade N in Hurtado et al. 2014) that excluded highly divergent taxa, which are more likely to produce homoplasies and complicate homology assessments. Alignments of the 12S rDNA, the 16S rDNA, and both genes concatenated, indicating included and excluded positions in subsequent analyses, are available as Supplementary fi le 1, Supplementary fi le 2 and Supplementary fi le 3. We used IQTree ver. 1.6.12 (Lam-Tung et al. 2015) to implement ModelFinder (Kalyaanamoorthy et al. 2017) and perform maximum likelihood (ML) analyses with standard bootstrap (100 replicates) and the Shimodaira-Hasegawa approximate Likelihood Ratio Test (SH-aLRT; 1000 replicates). We used MrBayes ver. 3.2.7a x86_64 (Altekar et al. 2004;Ronquist et al. 2012) as implemented in the CIPRES web portal (phylo.org) to perform Bayesian analyses (parameters in Dataset S1-3). We used PAUP* ver. 4.0a (build 168) (Swofford 2002) to compute pairwise uncorrected p-distances among taxa, for which all positions in the alignment were used, with missing or ambiguous positions ignored for each pairwise comparison.

Results
A total of 17 species of oniscideans belonging to nine families and 10 genera were identifi ed from Isla Grande. Of these, 16 were collected in coastal habitats (littoral and mangroves), two in Tropical Dry Forest, and fi ve in urban habitats (Table 1).
ANTENNA. Short and thickset, fl agellum of three articles, apical article as long as fi rst and second articles (Fig. 4A).

Remarks
Presently, the genus Tylos has a worldwide distribution and comprises 21 coastal species (Schmalfuss & Vergara 2000;Schmalfuss 2003). In Colombia, only T. niveus Budde-Lund, 1885 was recorded from the Caribbean region . Tylos negroi sp. nov. can be distinguished from all species of the genus by the shape of the ventral phylacomera 5. Moreover, it differs from T. niveus in the pereonite 1 epimeron having the inner lobe of the schisma surpassing the outer lobe (vs inner lobe not surpassing outer lobe) and the pereopod 7 propodus being infl ated (vs not infl ated).
Stenoniscus nestori López-Orozco, Taiti & Campos-Filho sp. nov. is morphologically similar to Stenoniscus carinatus, from which it differs mainly in the number and disposition of the dorsal ribs and tubercles on the cephalon and pereonites (compare Fig. 6A-B with Schmidt 2003: fi g. 67 and : fi g. 6a for S. carinatus). The new species is readily distinct from Stenoniscus pleonalis in having a strong dorsal ornamentation (vs very weak) and lacking the tomentose appearance due to long dorsal scale-setae (see Vandel 1944: fi g. 1a-c).

Distribution
Presently known only from Isla Grande, Cartagena de Indias, Colombia.

Remarks
Paoletti & Stinner (1989) described A. caraibicus from Parque Marrocoy, Falcón State, Venezuela. In the description, no sexual dimorphism was noted in the dorsal ornamentation. However, the male specimens show four dorsal tubercles on pleonite 2, while females have only two (see Fig. 9 and Paoletti & Stinner 1989: fi g. 11).
CEPHALON. Lateral lobes well developed and directed outwards, median lobe triangular, slightly surpassing distal margin of lateral lobes, frontal and suprantennal lines absent; eyes consisting of 5 ommatidia ( Fig. 13C-D).
ANTENNULA. Composed of two articles, second article bearing many lateral setae, distal margin bearing one fl agellar seta and two aesthetascs (Fig. 13F).
PLEOPODS. Pleopod 1 (Fig. 14D) exopod ovoid, wider than long; endopod stout, three times as long as exopod, apical portion slightly bent inwards. Pleopod 2 (Fig. 14E) exopod ovoid, wider than long; endopod with fl agelliform distal article. Exopods of pleopods 3-5 as in Fig. 14F-H. Schultz (1984) described A. ninae from San Pedro beach, Ambergris Cay, Belize. Comparing Shultz's description with the specimens examined here, it was possible to observe that almost all characters mentioned, including the pale dorsal pigmentation, are quite similar. However, Schultz (1984) mentioned that A. ninae has eyes composed of 14 ommatidia and antennula with three articles, and herein the specimens showed the eyes composed of 5 ommatidia and antennula of two articles. Most probably, the author misinterpreted the composition of the eyes of this species, since the illustrations of the cephalon clearly show a smaller number. The same statement can be applied to the antennula. In general, within Oniscidea, the composition of the antennula does not vary within the genera, with just few exceptions (see Schmidt 2002Schmidt , 2003. Moreover, analyzing other representatives of Armadilloniscus, it is possible to observe that the antennula is always composed of two articles (see Taiti & Ferrara 1989;Kwon & Wang 1996). Therefore, we identify our specimens as A. ninae.

Distribution
This species was previously recorded only from San Pedro beach, Ambergris Cay, Belize (Schultz 1984). First record for Colombia and Cuba.
ANTENNULA. Composed of three articles subequal in length, distal article with two lateral aesthetascs plus apical pair (Fig. 15F).
ANTENNA. When extended posteriorly reaches posterior margin of pereonite 5; fl agellum as long as fi fth article of peduncle, fi rst article slightly longer than second and third, second and third articles with three and two lateral aesthetascs, respectively (Fig. 15G).
PLEOPODS. Pleopod 1 (Fig. 17D) exopod triangular, inner margin almost straight, outer margin sinuous, distal portion triangular with distal margin rounded; endopod with thickest distal part with sides almost parallel, inner distal part with long pointed process concave on outer margin, prominent subquadrangular apical lobe on outer distal portion bearing thin setae. Pleopod 2 (Fig. 17E) exopod triangular, outer margin concave, bearing seven stout setae; endopod robust, distinctly longer than exopod. Exopods of pleopods 3-5 as in Fig. 17F-H. Vandel (1973) erected the genus Microphiloscia to allocate the new species M. trichoniscoides from Cueva de la Colorada, Provincia de Oriente, Sierra Maestra, Colorada del Maso, Cuba. After the examination of material belonging to this species, M. trichoniscoides shows the shape of the genital papilla and male pleopod 1 endopod typical of members of Halophiloscia (see also Taiti & Lopez 2008). Halophiloscia includes 9 halophilic species mainly distributed along the Mediterranean and Atlantic coasts of Europe and Africa (Schmalfuss 2003;Taiti & Lopez 2008;Taiti & Argano 2009. In America, only H. couchii (Kinahan, 1858) was recorded, from Argentina, Bermuda, and USA (see Schmidt 2003). The genus is mainly characterized by the runner-type habitus (sensu Schmalfuss 1984), epimera of pereonites 1-7 with one or more lines of noduli laterales, male pereopod 1 and sometimes pereopod 2 carpus and propodus enlarged and covered with several scales on frontal side, male genital papilla distally bifurcated, and male pleopod 1 endopod stout bearing a long, pointed process on the apex (see Schmidt 2003;Taiti & Lopez 2008). The species is included within the genus since it shows all the previously mentioned characters, except that the male pereopods 1 and 2 do not have the carpus and propodus enlarged. Thus, Microphiloscia is considered to be a junior synonym of Halophiloscia.

Distribution
Colombia. First record for continental islands of the Colombian Caribbean.

Distribution
Pantropical (Schmalfuss 2003). First record for continental islands of the Colombian Caribbean.

Distribution
Cosmopolitan species (Schmalfuss 2003). First record for continental islands of the Colombian Caribbean.

Additional reference
Campos .

Molecular phylogenetic analyses of Tylos Audouin, 1826
Sequences obtained for the three species of Tylos from Isla Grande and the specimen from Florida have been deposited under GenBank Accession Numbers MW532964-MW532970 and MW533069-MW533075. Only the 12S rDNA sequence was obtained for the specimen from Isla Grande assigned to T. marcuzzii. Phylogenetic analyses using only 12S rDNA group this sequence with the two specimens of T. marcuzzii from Cuba reported in Hurtado et al. (2014); see Fig. 18. The 12S rDNA p-distance between T. marcuzzii from Isla Grande and T. marcuzzii from Cuba is 2.0-2.7%, whereas the distance between the specimens from the two Cuban localities is 2.0% (Table 2). Only the 16S rDNA sequence was obtained for the specimen of T. niveus from the Florida Keys, the type locality of this species. Phylogenetic analyses of 16S rDNA show that specimens identifi ed as T. niveus from Isla Grande are most closely related to the T. niveus specimen from the Florida Keys. The 16S rDNA p-distance between T. niveus from Isla Grande and the Florida Keys is 2.7% ( Table 2).
The concatenated (12S rDNA and 16S rDNA) dataset was comprised of 43 taxa and 740 characters (excluding characters with ambiguous homology), 236 of which were parsimony-informative. The specimens assigned to T. marcuzzii were used as outgroup. Five main lineages were identifi ed for the ingroup taxa in the concatenated phylogenetic analyses, which formed a basal polytomy (Fig. 18) (Table 1).
Molecular phylogenetic analyses supported the identifi cation of three species of Tylos in Isla Grande and the recognition of one of them as a new species. The species identifi ed as T. marcuzzii clustered with specimens identifi ed as this species from two Cuban localities. The genetic distance between the specimens from Isla Grande and those from Cuba ranged between 2.0 and 2.7% for 12S rDNA, whereas the divergence between the two Cuban specimens was 2.0%. These probably correspond to species-level divergences . The Isla Grande specimens from Laguna Caracolí and Caño Ratón recognized as T. niveus were recovered as closely related with the specimen from the Florida Keys, type locality of the species. The genetic distance between specimens of T. niveus from the Isla Grande and the Florida Keys was 2.7% for 16S rDNA. The lineage formed by the T. niveus from Isla Grande and Florida is part of a cluster in which a basal polytomy of fi ve lineages, including this lineage, was observed. Another basal lineage corresponds to the new species T. negroi sp. nov., indicating the high degree of genetic differentiation of this isopod within this cluster. The other three basal lineages include a clade of specimens from the Northeastern Pacifi c (referred to as T. punctatus sensu lato in Hurtado et al. 2014), which appears to be a clade conformed by multiple species ), a lineage previously identifi ed as T. niveus from Aguada, Puerto Rico , and a lineage previously identifi ed as Tylos sp. from Yaguanabo, Cuba . Genetic distances among the basal lineages range from 9.7 to 15.5% for 12S rDNA and 11.1 to 13.6% for 16S rDNA, which appear to correspond to species-level divergences in Tylos. Hurtado et al. (2013) reported smaller genetic divergences among multiple lineages that show morphological differences and appear to represent different species within the Northeastern Pacifi c clade. Although the lineage from Puerto Rico was previously identifi ed as T. niveus, it probably corresponds to a different species, because it is highly divergent from the specimen from the Florida Keys, type locality of T. niveus. Similarly, Tylos sp. from Yaguanabo, Cuba, also appears to correspond to a different species. It is important to obtain more specimens of Tylos from the Caribbean to better understand the diversity patterns of this group in the region, and to use more markers, employing genomic methods, which could help solve the relationships among the fi ve basal lineages.
We identifi ed the Ligia found in Isla Grande as L. baudiniana. The taxonomy of L. baudiniana, however, needs revision. This species was originally described by Milne-Edwards (1840), from the surrounding of the San Juan de Ulúa Fort in the city of Veracruz, Gulf of Mexico, Mexico. However, specimens recently collected by LH from there corresponded to Ligia exotica Roux, 1828 (Santamaria et al. 2014;Hurtado et al. 2018). Santamaria et al. (2014) identifi ed a monophyletic clade of Ligia distributed in the Caribbean Sea, Bahamas, southern Florida, Bermuda, and the Pacifi c coast of Central America and Colombia. Males of this clade have in their pleopod 2 endopod a large lateral process that bifurcates close to the apex (Leistikow 1997: fi g. 6 and Santamaria et al. 2014: fi g. 4). Schultz (1972) and Schultz & Johnson (1984) assigned specimens from Florida and Bermuda with this characteristic to L. baudiniana. Santamaria et al. (2014) found that this clade is comprised of seven highly divergent lineages, which, based on their divergences, might warrant separate species status. One of the clades (Clade E in Santamaria et al. 2014), was observed on the Magdalena coast of Colombia, about 280 km northeast of Isla Grande. Unfortunately, we could not obtain molecular data for the specimens of Isla Grande, but molecular analyses will reveal whether they also belong to Clade E or to another lineage.
This study clearly shows the high potential for discovering unknown biodiversity of coastal oniscideans, a poorly studied group, in the Caribbean region. An examination of the oniscidean diversity on a tiny island of the Colombian Caribbean resulted in the description of three new species of coastal oniscideans. This and previous phylogeographic studies of isopods of Tylos and Ligia are revealing high levels of cryptic biodiversity in the Caribbean region, suggesting a high potential for fi nding much more diversity if sampling is expanded Santamaria et al. 2014). As coastal habitats are threatened by multiple factors in the region, and unique evolutionary lineages of coastal oniscideans are at imminent risk, it is important to continue taxonomic and systematic studies of this group to identify diversity patterns and protect members of this highly diverse group. Coastal isopods can be informative on the processes that have contributed to the extraordinary diversity of the Caribbean region. For example, the phylogeographic study of Ligia allowed for testing different hypotheses to explain diversifi cation patterns within this group, which included competing geological hypotheses and oceanic circulation patterns, and also provided evidence of multiple Atlantic-Pacifi c divergences, which appear to refl ect separate vicariant events (Santamaria et al. 2014). Interestingly, studies of other coastal isopods have also shown multiple transisthmian divergences, a highly debated topic in evolutionary studies of the region (Hurtado et al. 2016. Coastal oniscideans also have the potential of being used as biomonitors of contamination (García-Hernández et al. 2015), and it is important to understand their diversity well in order to correctly interpret coastal ecological studies (Mattos et al. 2018). Our work on Tylos highlights the importance of combining taxonomic and molecular analyses to support taxonomic decisions and uncover cryptic diversity. The already published dataset of mitochondrial sequences for most nominal species of this genus ) made the molecular phylogenetics analyses for specimens of Tylos possible. Given the multiple threats to coastal habitats in the Caribbean region, it is of upmost importance to generate similar molecular information for other oniscidean species to better analyze their diversifi cation patterns in the region.