Nine species of Parasesarma are currently recorded from continental Australian mangroves. The present study describes a new species, P. otiense sp. nov., from Western Australia. Parasesarma otiense sp. nov. occurs sympatrically with P. hartogi and P. holthuisi in the mangroves of Exmouth Gulf. In both COX1 and 16S BI trees, the new species is well nested within Parasesarma, but divergent from other species. Morphologically, the new species is distinct from other Parasesarma in having eight asymmetric tubercles with the distal slope longer than the proximal slope on the upper surface of the cheliped dactylus. Parasesarma otiense is the tenth species of Parasesarma recorded from continental Australia, taking the number of species assigned to the genus Parasesarma to 59. A key to the species of Parasesarma known from Australian waters is provided to aid in their identification.

Crustacea, COX1, mangrove crab, molecular taxonomy, morphology, Parasesarma otiense

Brachyuran crabs of the genus Parasesarma De Man, 1895 are among the most common components of mangroves and estuaries (Lee 1998, 2015) and this genus has been the subject of significant recent taxonomic research. Based on molecular and morphological analyses, Shahdadi and Schubart (2018) transferred most species of Perisesarma De Man, 1895 to Parasesarma, and Shahdadi et al. (2020) transferred most of the long-legged species of Parasesarma to a new genus Leptarma Shahdadi, Fratini & Schubart, 2020. Currently, Parasesarma includes 58 species distributed across the Indo-West Pacific, with highest species diversity in Southeast Asia (Shahdadi et al. 2018, 2020, 2023).

Nine species of Parasesarma have been recorded from continental Australian mangrove habitats, with approximately half of the species endemic to either the eastern or western coasts, with some potential overlap zones along the northern coast between the Kimberley region of Western Australia to the Gulf of Carpentaria and eastern Cape York Peninsula in Queensland (Davie 1985). Parasesarma lividum (A. Milne-Edwards, 1869), P. brevicristatum (Campbell, 1967), and P. erythodactyla (Hess, 1865) have been recorded from the east coast (Campbell 1967; Davie 1993; Shahdadi et al. 2019). Parasesarma longicristatum (Campbell, 1967) and P. messa (Campbell, 1967) have a wider distribution from the east to the north coasts (Campbell 1967; Shahdadi et al. 2018). Parasesarma darwinense (Campbell, 1967), P. austrawati Shahdadi, Davie & Schubart, 2019, P. holthuisi (Davie, 2010), and P. hartogi Davie & Pabriks, 2010 have been recorded from northern to western coasts (Campbell 1967; Davie 2010; Davie and Pabriks 2010; Shahdadi et al. 2019). In addition to these species, P. sigillatum (Tweedie, 1950) is endemic to the Australian Indian Ocean Territory of the Cocos (Keeling) Islands (Ng et al. 2016).

The integrated molecular and morphological analyses of recent studies (e.g. Shahdadi et al. 2018, 2020; Shih et al. 2023) have provided a framework for new taxonomic research by clarifying the placement and the morphological delimitation of many species that were described during the 19^th and early 20^th centuries. The present study uses sequence data from specimens across a range of species and integrates this with morphology to describe a previously unknown species.

Specimens were collected during different expeditions conducted by the Western Australian Museum: NCB Exmouth Muirons in 2016, Bush Blitz Cape Range in 2019, Environs Kimberley Broome in 2023, and Kimberley Reef Connect in 2023. Specimens were collected by hand from different localities (see material examined and Suppl. material 1) along the intertidal fringing mangroves and associated mud flats during low tides. The specimens were fixed in 96% ethanol and preserved in 75% ethanol. They were transferred to the Western Australian Museum (WAM), Perth, for further morphological examination and tissue subsampling for DNA extraction. All specimens are housed in the WAM or the Biodiversity Research Museum, Biodiversity Research Center, Academia Sinica, Taiwan (ASIZ).

Abbreviations used are as follows: bp: base-pairs; coll.: collected; cl: carapace length along the midline; cw: maximum carapace width; P2–P5: pereiopods 2–5, respectively (first to fourth ambulatory legs, respectively); G1: male first gonopod. Measurements are in millimetres (mm). The description is based on the holotype male with ranges and variations given in parentheses for paratypes, followed by female specific characters observed in the female paratype.

Genomic DNA was isolated from muscle tissue using the Qiagen DNeasy extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. A partial segment of the mitochondrial protein-coding gene cytochrome c oxidase subunit 1 (COX1), corresponding to the barcode region (Hebert et al. 2003), and a partial segment of 16S ribosomal DNA (16S) were selected as the most commonly used genetic markers in species delimitation in Parasesarma (e.g. Shahdadi et al. 2018). The polymerase chain reactions (PCRs) were performed using dgLCO1490 5′-GGTCAACAAATCATAAAGAYATYGG-3′ as forward primer and dgHCO2198 5′-TAAACTTCAGGGTGACCAAARAAYCA-3′ as reverse primer (Meyer 2003) for COX1; and 16L29 5′-YGCCTGTTTATCAAAAACAT-3′ as forward primer and 6H11 5′-6H11 AGATAGAAACCRACCTGG-3′ as reverse primer (Schubart 2009) for 16S. The PCR reactions were performed using 12.5 μl ThermoScientific DreamTaq Green PCR Master Mix (2×), 0.75 μl of each primer (10 μM), 1 μl of template DNA, and 10 μl of distilled water. The PCRs were conducted in a DNA Thermal Cycler T100 (Bio-Rad, Richmond, CA, USA) with the following profiles: an initial 7 cycles of 25 s at 95 °C, 25 s at 52 °C and 40 s at 72 °C; followed by 35 cycles of 25 s at 95 °C and annealing for 25 s at 46 °C for COI and 48 °C for 16S, 40 s at 70 °C for extension; and a final extension step of 7 min at 72 °C. New sequences were submitted to GenBank (https://www.ncbi.nlm.nih.gov/) and are available under accession numbers (Table 1). Sequences were assembled, proofread, and the primer regions were removed using Geneious Prime (https://www.geneious.com). Further quality control was undertaken through checking for stop codons in the translated sequence and checking for matches with non-target taxa on Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences of other Parasesarma were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and used for phylogenetic analyses. Sesarmoides longipes (Krauss, 1843), Fasciarma fasciatum (Lanchester, 1900), and Perisesarma dussumieri (H. Milne Edwards, 1853) were used as outgroups (see phylogenetic trees in Shahdadi et al. 2018, 2020) (for the accession numbers see Figs 1, 2, the phylogenetic trees). The sequences were aligned with ClustalW (Thompson et al. 1994) implemented in BioEdit 7.0.5 (Hall 1999).

Figure 1. 

Bayesian Inference phylogram constructed in BEAST 2.7.7 for COX1 sequences of Parasesarma. The numbers behind the nodes refer to the support values (posterior probability) (posterior probabilities under 0.90 are not shown). Sequences belonging to Sesarmoides longipes, Fasciarma fasciatum, and Perisesarma dussumieri were used as outgroups. The numbers in front of species names are GenBank accession numbers. Sequences obtained in the present study are shown in red and the newly described species are in bold font (the numbers left to species names are Western Australian Museum numbers).

Figure 2. 

Bayesian Inference phylogram constructed in BEAST 2.7.7 for 16S sequences of Parasesarma. The numbers behind the nodes refer to the support values (posterior probability) (posterior probabilities under 0.90 are not shown). Sequences belonging to Sesarmoides longipes, Fasciarma fasciatum, and Perisesarma dussumieri were used as outgroups. The numbers in front of species names are GenBank accession numbers. Sequences obtained in the present study are shown in red and the newly described species are in bold font (the numbers left to species names are Western Australian Museum numbers).

Available sequences of COX1 cover more species of Parasesarma, compared to the available sequences of 16S. We, therefore, conducted phylogenetic analysis for each gene separately. To address the phylogenetic positions of the new material, a Bayesian Inference (BI) was conducted in BEAST 2.7.7 (Drummond and Rambaut 2007) for each gene. We used the Yule Model (as prior for tree model) and a strict clock model. Markov Chains were run for 10 million generations, sampling every 1,000 iterations and discarding the first 10% as burn-in. The remaining 9,000 trees were used to calculate the maximum clade credibility tree in Tree Annotator (part of the BEAST package). The best evolutionary models were TIM2+F+I+G4 for COX1, and TIM+F+I+G4 for 16S as determined by using ModelFinder (Kalyaanamoorthy et al. 2017) through the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/?user=guest&jobid=241119083607) (Trifinopoulos et al. 2016) based on BIC scores. To calculate genetic distances (p-distance) we used MEGA X (Kumar et al. 2018).

Parasesarma otiense sp. nov. co-occurs with P. hartogi and P. holthuisi (see comparative material listed in Suppl. material 1) within the Bay of Rest but differs in carapace morphology, with no sign of epibranchial teeth (Fig. 3C). In contrast, P. hartogi (see Davie and Pabriks 2010: fig. 1B) has a distinct epibranchial projection and P. holthuisi (see Davie 2010: fig. 1B) has a small but clearly incised epibranchial tooth. The cheliped dactylus in P. otiense sp. nov. is straight with eight distinct rounded, asymmetric tubercles (Fig. 3E–G), rather than downcurved with low tubercles in P. hartogi (see Davie and Pabriks 2010: fig. 2A) or with 12 or 13 transversely broadened tubercles (Davie 2010: fig. 2B) in P. holthuisi.

Of the eight other species found in Australian territories, P. austrawati, P. brevicristatum, P. darwinense, P. lividum, P. longicristatum, and P. messa all bear a well-developed epibranchial tooth on each side of the carapace (Campbell 1967; Shahdadi et al. 2018, 2019). While P. erythodactyla from eastern Australia (Davie 1993) and P. sigillatum from the Cocos (Keeling) Islands (Ng et al. 2016) lack epibranchial teeth but can be distinguished from P. otiense sp. nov. by the number and morphology of the tubercles on the cheliped dactylus. Parasesarma erythodactyla has 23–26 symmetrical tubercles (see Davie 1993: fig. 3A, C), and P. sigillatum has 16–18 triangular tubercles (see Ng et al. 2016: fig. 3E–H), compared with only eight asymmetric tubercles in P. otiense sp. nov. (Fig. 3E–G).

Parasesarma ellenae (Pretzmann, 1968) is distributed in the South Pacific islands and has six or seven rounded asymmetric tubercles on cheliped dactylus (Shahdadi et al. 2021). Parasesarma kuekenthali (De Man, 1902) has eight or nine asymmetric tubercles on the cheliped dactylus and occurs in Southeast Asia from the Philippines to the Indonesian islands of Sulawesi and Halmahera (Shahdadi et al. 2021). The tubercles in these two species are, however, different from the new species as the proximal slope is longer than the distal slope in P. ellenae (see Shahdadi et al. 2021: fig. 9E) and P. kuekenthali (see Shahdadi et al. 2021: fig. 14D), whereas in P. otiense sp. nov. the distal slope is longer (Fig. 3E).

Understanding the diversity of Australian Parasesarma has been hindered by a complicated taxonomic history of the group (Shahdadi et al. 2018). Molecular phylogenetics has supported the distinction of many morphologically similar or cryptic species and has clarified some characters as well as distributions. In the case of P. otiense sp. nov., the phylogenetic results (both COX1 and 16S) support the morphological differences observed (Figs 1, 2). The genetic distances between the new species and the closest species in both trees are much higher than the distances between previously known species pairs of Parasesarma (Shih et al. 2023) as well as many other crustacean sister species (Matzen da Silva et al. 2011; Chu et al. 2015).

While not central to this study, the identification of P. longicristatum from Exmouth Gulf represents a considerable extension of the known range south from Admiral Island in the Kimberley region. Previously, the known range of P. longicristatum extended from Moreton Bay, Queensland, across northern Australia, westwards into the Kimberley region of Western Australia, and this new record makes this species the most widespread member of the genus in Australia (Shahdadi et al. 2018).

Although identifying species within the genus is not a trivial endeavour, the key below provides clear and relatively simple steps to aid in the identification of the known Australian Parasesarma.

Specimens examined in this study were collected during several projects organised and/or funded by the Bush Blitz Species Discovery Program, Gorgon Net Conservation Benefits Fund (NCB), Environs Kimberley and Kimberley Reef Connect. We wish to thank the Bush Blitz team Jo Harding, Haylee Weaver, Kate Grarock, and Zoe Jarvis for running the Bush Blitz Cape Range 2019 Expedition, a partnership project between the Australian Government, BHP and Earthwatch Australia; Nerida Wilson and Clay Bryce (both formerly at WAM) for organising the NCB Exmouth Gulf and Muiron Islands 2016 Expedition. Zoe Richards (WAM and Curtin University), Tom Vigilante and the Wunambal Gaambera rangers for running and being a part of the Kimberley Reef Connect project funded by Parks Australia; Victoria De Bruyn (formerly of Environs Kimberley) and Bart Pigram for their assistance in the field and access to mangroves around Broome and Barred Creek. The new DNA sequences were generated through the WAM Genetic Resources laboratory. The authors would like to contribute this species to the Ocean Census Programme (https://oceancensus.org/). This is Ocean Census Species Number 200.