Beyond shells: first detailed morphological description of the mangrove-associated gastropod Haminoea cf. fusca (A. Adams, 1850) (Cephalaspidea, Haminoeidae), with a COI phylogenetic analysis

ABSTRACT The diversity of Haminoea Turton & Kingston, 1830 snails is poorly understood in the Indo-West Pacific. These gastropods occur in shallow subtidal and intertidal areas usually associated with algae, seagrass, or coral reefs, and one species, often identified as Haminoea fusca (A. Adams, 1850) is regarded to be restricted to mangrove habitats. In this paper we provide the first detailed description of this species by means of anatomical dissections, scanning electron microscopy, and DNA barcodes. A Bayesian COI gene tree including specimens from Pakistan and the Philippines together with other Indo-Pacific and Atlantic lineages was inferred and the possible existence of more than one species under the name H. fusca is highlighted and discussed. In Pakistan Haminoea cf. fusca was found to inhabit tidal estuarine mud-flats with oyster reefs.


INTRODUCTION
and Sandspit near Karachi in their study of mangrove associated molluscs. The specimens were found to favour elevated substrates of sand and clay, and occurred throughout the tidal zone, even crawling up the mangrove pneumatophores. However, this name is a junior synonym of the well-known and economically important species Bullacta caurina (Benson, 1842) (synonym of Bullacta exarata (Philippi, 1849)), which is endemic to South Korea and China (Tchang 1934;Habe 1952;Burn & Thompson 1998;Higo et al. 1999;Malaquias 2010).
In this study, we confirm the occurrence of Haminoea cf. fusca (A. Adams, 1850) in Pakistan associated with intertidal mud flats with oyster reefs and we provide for the first time a detailed morphological characterization of the species by means of fine anatomical dissections and scanning electron microscopy. A COI molecular phylogeny is inferred to aid in species delimitation and to preliminary address species relationships.

Taxa sampling
Specimens of H. cf. fusca were collected during biodiversity assessments from intertidal oyster reefs in estuarine muddy areas on the Balochistan Coast in Pakistan. The sampling site is on the Sindh Province side of the mouth of the Hab River Delta (24°53'12.01''N, 66°42'14.00''E; also spelled Hub River Delta). Live animals were photographed, relaxed by freezing in sea water and preserved in 95% ethanol. Specimens were sent for study and are housed at the Invertebrate Collections, Department of Natural History, University Museum of Bergen, University of Bergen (voucher number: ZMBN 125424). Additional specimens and egg masses are stored at the Centre of Excellence in Marine Biology (Study collection of Sadar Aslam, Pakistan). For the molecular phylogenetic analysis 34 COI sequences of Haminoea from the Atlantic, East Pacific and Indo-West Pacific were obtained from GenBank (Table 1). The tree was rooted with the haminoeid Atys jeffreysi (Table 1; Fig. 1).
morpho-anaTomical work Shells, male reproductive systems and anterior digestive system were dissected out of the animals. Shells were imaged with a DSLR camera equipped with macrolens. The male reproductive system was drawn using a stereo microscope fitted with a drawing tube. The anterior digestive system was dissected and the gizzard and buccal bulb were dissolved in a solution containing 180 µl buffer ATL with 20 µl of proteinase K-solution incubated at 56°C for approximately 4-6 hours (protocol modified from Holznagel [1998] and Vogler [2013]) [buffer and enzymes were obtained from the Qiagen DNeasy® Blood and Tissue Kit, catalogue no. 69504. The gizzard plates were critical-point dried after cleaning to maintain natural shape. The radulae and gizzard plates were mounted on metallic stubs using carbon sticky tabs and coated with gold-palladium. The samples were scanned and imaged with a Zeiss Supra 55VP scanning electron microscope.
Dna ExTracTion, amplificaTion anD sEquEncing DNA was extracted from foot tissue of two specimens using the Qiagen Dneasy® Blood and Tissue Kit (Catalogue No 69504) following the protocol recommended by the manufacturer. Barcodes consist of partial sequences (c. 640 bp) of the mitochondrial gene cytochrome c oxidase subunit I (COI) and were amplified using the universal primers designed by Folmer et al. (1994). Polymerase chain reactions (PCR) were performed following the protocol described by Malaquias et al. (2009) using Qiagen Taq DNA polymerase. Successful PCR products were purified according to the EXO-SAP protocol described by Eilertsen & Malaquias (2013). The purified products were sequenced using an ABI 3730XL DNA Analyser (Applied Biosystems). The generated sequences were deposited in GeneBank (Table 1).   et al. 1995;Natterer & Neumann 2013). The in-group was formed by 36 sequences of Haminoea spp. and the tree was rooted with the haminoeid species Atys jeffreysi (Weinkauff, 1866). Uncorrected p-distances were calculated in MEGA 7.

phylogEnETic analysEs
Both analyses resulted in similar tree topologies, but the tree inferred from the dataset including the 3 rd codon positions was better resolved with higher node supports ( Fig. 1, Appendix). This latter analysis suggests a possible 18 th putative species (Fig. 1). The specimens of Haminoea cf. fusca from Pakistan clustered together with a specimen of H. fusca from the Philippines with maximum support. The COI uncorrected p-distance between the Pakistani and the Philippine specimens varied between 7.3-7.5% and between the two Pakistani specimens was 0.2%. The clade with all specimens of H. fusca was the sister group of a clade (  Haloa vitrea -Hung 2013. Haminoea tenera -Mujiono 2016: 47, fig. 4a Gizzard plates (Fig. 3D-F) Flat surface, ridges absent. Rachis present. Surface covered in small tightly arranged pointed rods; rods tapering in size outwardly; larger worn rods on top of rachis.
Male reproductive system (Fig. 4A, 1965). The fundus has thick walls forming folds resembling a penial papilla (Fig. 4B). The limited research available on the genus Haminoea and ambiguous original descriptions of most IWP species often hampered authors to identify correctly the species. For example, Strack (1998 Yonow & Jensen (2018) have recently described the species H. edmundsi from Ambon, Indonesia based on features of the external morphology, colouration, and shells (no anatomical details were included). The authors claim that the animal did not resemble any extant species of Haminoea, but the image included in the work is a perfect match with live images of H. fusca (Gosliner et al. 2008;2015;Hung 2013;Riek 2013, 2014, Mujiono 2016Cobb 2018), and thus we here consider that H. edmundsi could be a junior synonym of H. fusca. However, the genetic distance found between the specimens from Pakistan and the one from the Philippines (COI uncorrected p-distance = 7.3-7.5%;   (1901: pl. 18, figs 44, 47, pl. 19, figs 4, 5), detail of radula with "a": central rachidian (pl. 18, fig. 44 fig. 94) was based on shells, which the author described as ovo-globose, covered in fine spiral striae, with dark periostracum and internally brownish. A. Adams (1850)' shells are similar to those from Pakistan; however, the latter ones are whitish-brown with a pale orange periostracum (Fig. 2B), and the specimen from the Philippines (here coded "C37"; Fig. 1; Table 1) and illustrated by Gosliner et al. (2008Gosliner et al. ( , 2015 has a completely white shell with an apparent translucent periostracum. Variability was also found in the COI gene with specimens from Pakistan and the Philippines (C37) varying between 7.3-7.5%, a range in Cephalaspidea gastropods, considered sufficient to indicate that lineages have speciated (Eilertsen & Malaquias 2013;Ohnheiser & Malaquias 2013;Malaquias et al. 2016).
This genetic variability together with the limited original description of H. fusca and the apparent variable features found in this species render its identity questionable and raise the possibility that more than one species of similar ecological requirements associated with mangroves and mud-flat habitats may in fact occur across IWP. Although, this can only be tested with a thorough study of the morphological and genetic diversity of the species including representatives from across its entire geographical distribution. Nevertheless, and since the type locality of the species is the Philippines, we cautiously refer to the Pakistani specimens as Haminoea cf. fusca.
The only known anatomical study of H. fusca was performed by Bergh (1901) based on preserved specimens from Macau. Bergh (1901) did not figure the animal or shell, but mentioned that the cephalic shield had a posterior shallow indentation. Additionally, he included detailed drawings of the radula, gizzard plate and male reproductive system (Fig. 2C). The drawings show a rachidian of similar shape to those of our specimens with rounded cusps, ridge-less gizzard plates covered in small rods and a compact male reproductive system with a lateral bulge and a bulbous prostate apparently continuous with the seminal duct. These similarities with our specimens suggest conspecificity between the samples from Pakistan and those studied by Bergh (1901) from the South China Sea (Macau).
The majority of Haminoea species live in shallow marine waters and H. fusca is the only intertidal mangrove-associated species of the genus (Strack 1998;Ng & Sivasothi 2001;Lozouet & Plaziat 2008;Hung 2013;Riek 2013Riek , 2014Mujiono 2016;Cobb 2018;Yonow & Jensen 2018). It seems that this species has the capacity to crawl up the pneumatophore of the mangroves and tolerate periods of emersion (Ullah et al. 2018) and during our fieldwork campaigns specimens were observed outside water among intertidal oysters during low tide. In the Hab River Delta around Sonari (Sunhera) Beach (24°52'56.69''N, 66°41'12.77''E), nearby our sampling site, there used to be lush mangrove forests that disappeared after the construction of the Hab River's dam has reduced the volume of freshwater with devastating consequences for the local mangrove habitats (Khan 1979). The average salinity in the area is now 34 parts per thousand which has been also causing decline of the oyster reefs and their associated ecosystems. Despite this fact, H. cf. fusca is, even in the absence of mangroves, still present and reproducing in the area.