Review and integrative taxonomy of the genus Prosopistoma Latreille, 1833 (Ephemeroptera, Prosopistomatidae) in Thailand, with description of a new species

Abstract Three species of the genus Prosopistoma Latreille, 1833 (Prosopistomatidae) are currently reported from Thailand. A new species, Prosopistomacarinatumsp. n., is described here based on specimens from western and southern Thailand. The new species can be easily distinguished from the other members of Prosopistoma by the following combination of characteristics: (i) the presence of two ridged longitudinal lines on each side of its carapace, (ii) antenna 7-segmented, (iii) a strongly convex carapace and (iv) nine pectinate setae on the ventral margin of the fore tibiae. A comparison between the key characteristics of P.carinatumsp. n. and the known Thai species is provided. Results of analysis of the mitochondrial cytochrome oxidase I (COI) gene (658 bp) of three species, as well as the distribution of the Thai species, are also discussed.


Introduction
Prosopistomatidae (Ephemeroptera) is a monogeneric family, represented by the genus Prosopistoma, which was originally established by Latreille (1833). Of the 27 species described to date, 16 species have been described from the Oriental region (Lieftinck 1932, Soldán and Braasch 1984, Tong and Dudgeon 2000, Sartori and Gattolliat 2003, Zhou and Zheng 2004, Barber-James et al. 2008, Barber-James 2009, Shi and Tong 2013Balachandran et al. 2016, Roopa et al. 2017). An updated key to the known species of this realm was provided by Shi and Tong (2013).
In Thailand, only the larvae of Prosopistoma annamense Soldán & Braasch, 1984, P. sinense Dudgeon, 2000, andP. wouterae Lieftinck, 1932 are known (Parnrong et al. 2002, Tungpairojwong andBoonsoong 2011). In the present study, we describe a new species of Prosopistoma based on specimens from western and southern Thailand. In addition, a distribution map of the genus in Thailand and mitochondrial COI sequence data are provided.

Materials and methods
The prosopistomatid mayfly larvae were collected from streams and rivers in northern, north-eastern, eastern, central, western, and southern Thailand from 2013 to 2018. Measurements (given in mm) and photographs were taken using a Visionary LK System (Dun, Incorporated, USA). All drawings were made with the aid of a camera lucida attached to a compound microscope. For Scanning electron microscopy (SEM), specimens (head, carapace, sternal plate, foreleg) were dried in a Critical Point Drier (CPD7501) and coated with gold (10 nm, Sputter Coater SC7620). SEM photographs were observed by a FEI Quanta 450 SEM. Final plates were prepared with Adobe Photoshop CC 2017. The material is deposited in the collection of the Zoological Museum at Kasetsart University in Bangkok, Thailand (ZMKU) and at the Museum of Zoology in Lausanne, Switzerland (MZL). The distribution map was generated via the Simple Mapper website (http://www.simplemappr.net) using GPS coordinates.
The collected specimens were fixed in absolute ethanol and preserved under refrigeration for description and DNA extraction. Details of the specimens from the three species used for the DNA experiment are shown in Table 1. Total DNA extraction was performed using a genomic DNA purification kit (NucleoSpin, Macherey-Nagel, Germany) following the manufacturer's protocol. A fragment of the mitochondrial gene cytochrome oxidase I (COI) gene was amplified (658 bp) using the primers LCO1490 (5'-GGT CAA ATC ATA AAG ATA TTG G-3') and HCO2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3'), designed by Folmer et al. (1994). Polymerase chain reaction (PCR) conditions were as follows: a 50 μl final reaction volume containing 25 μl of PCR Master mix solution, 1 μl (10 μm) of each primer, 2 μl of total DNA and 21 μl of nuclease free sterile water. PCR was performed as follows: 5 minutes at 94 °C, then 30 seconds at 94 °C, 30 seconds at 48 °C, and 60 seconds at 72 °C (40 cycles), and a final elongation step at 72°C for 10 minutes (Gattolliat et al. 2015). Pu-rification and sequencing were conducted by Macrogen, Inc. (South Korea). Sequence alignment and editing were performed using ClustalW. The best-fit evolution model obtained was T92 (Tamura 3-parameter) + G. Phylogenetic trees based on maximum likelihood (ML) were performed with MEGA 7 using the likelihood-ratchet method with 1,000 bootstrap replicates. Pairwise (uncorrected-p) sequence distances were also calculated using MEGA 7 (Kumar et al. 2016). Nucleotide sequences obtained in this study have been deposited in the GenBank database (MK285321-MK285330).   Diagnosis. The larvae of P. annamense can be distinguished from those of other Oriental congeners by (i) apex of the inner margin of the fore tibiae, with 4-6 serrated pectinate spines (Fig. 3B), (ii) three long, finely serrated bristles at the base of the inner incisor, on both right and left mandible, (iii) segment III of the maxillary palp slightly shorter than 1/3 the length of segment II and (iv) posterolateral spines at segments VII and VII parallel to or bent outwards from the body axis ( Fig. 1A, B).
Remarks. The larvae of P. annamense were originally described by Soldán and Braasch (1984) and collected from Vietnam (Thuan Hai province). They are widely distributed in southern and central Vietnam. Tungpairojwong and Boonsoong (2011) reported this species in middle and moderately disturbed streams in Loei province (NE), Chaiyaphum province (NE) and Kanchanaburi province (W). In this study, we found this species in several provinces, and it seems to have a wide distribution in Thailand. Surprisingly, we found larvae on the banks of the Khwae Noi River (Kanchanaburi province) (Fig. 12C). This is the first report of Prosopistoma larvae in large rivers in Thailand, and the larvae collected from large rivers are paler than those larvae from streams (Fig. 1B).  Diagnosis. The larvae of Prosopisoma sinense can be distinguished from those of other congeners by (i) antennae with 4-5 segments, (ii) antenna segment III much longer than the combined length of segments IV-V (iii) and apex of the ventral margin of the fore-tibia with 6-8 serrated spines (Fig. 4B) (Shi and Tong 2013).
Remarks. The larvae of P. sinense were originally described by Tong and Dudgeon (2000) and collected from China (Guangdong, Hongkong). In Thailand, Tungpairojwong and Boonsoong (2011) reported this species from a slightly disturbed stream in Kanchanaburi province. In this study, we found the larvae in the same habitat (Fig. 12D) and streams as previous studies. In addition, we found this species at a slightly disturbed stream in Chiang Dao, Chiang Mai province (N). Figure 14 Prosopistoma wouterae Lieftinck, 1932: 44-55, pls 1-2. (orig.); Lafon, 1952: 433 (  Diagnosis. The larvae of P. wouterae can be distinguished from those of other congeners by the combination of the following characteristics: (i) apical segment of the maxillary palp shorter than 1/2 the length of segment II, (ii) mesonotum with five dark-brown patches connected by brown narrow stripes, (iii) apex of the ventral margin of the fore-tibiae with eight serrated spines and (iv) posterolateral spine of segments VII and VIII parallel to or bent inwards the body axis (Shi and Tong 2013).
Remarks. Prosopistoma wouterae was originally described by Lieftinck (1932) from West Java. Parnrong et al. (2002) reported larvae of P. wouterae from the Songkhla province. In this study, we sampled in the same stream (Boripat Waterfall) but unfortunately, no specimens were found during our collection. This species seems to have a distribution limited only to the Sunda Islands and southern Thailand. Only four genes (16S, 18S, 28S rDNA, and H3) were sequenced for P. wouterae (Ogden and Whiting 2005), and no COI sequences were available for comparison.
Diagnosis. The larvae of Prosopistoma carinatum sp. n. can be distinguished from those of other species by the combination of the following characteristics (Table 3): (i) carapace with two longitudinal ridges on each side of the midline on its surface (Fig. 1F), (ii) antenna 7-segmented, (iii) carapace with three pale-coloured depressions on each side, (iv) carapace with a very narrow flange width and strong convexity, (v) carapace with a typically brown colouration and a distally irregular (zig-zag) yellow pattern at 0.7 times the length of the carapace, (vi) carapace with circular scale-like structures, (vii) presence of three long serrated bristles at the base of the inner canine and (viii) ventral margin of the fore-tibia with nine pectinate setae.
Description (in alcohol). Body length 1.94-2.43 mm excluding caudal filaments. Head. Head yellowish with median blackish ocellus between antennae (Fig. 8A), head width approximately 3 times length. Epicranial ecdysial sutures prominent, passing through the anterior margin of the lateral ocelli, and between the compound eyes and the antennal bases and continuing to the lateral margin of the head (Fig. 8B). Antenna 7-segments (Figs 6A, 8B, 8C), longer than the distance from antennal base to anterior margin of head; segment I usually retracts into head capsule; segment III the longest and shorter than combined length of segments IV-VII, antenna segment VII minute (Fig. 8C). Labrum (Fig. 6D) narrow, 3 times wider than long, anterior    (Soldán and Braasch 1984, Sartori and Gattolliat 2003, Shi and Tong 2013    margin fringed with dense fine setae. Left and right mandibles similar (Fig. 6E, 6F). Outer canine of mandibles subequal and broader than inner canine, with three apical teeth, a smaller outer tooth and a larger inner tooth with a serrated margin near the apex and three small spines; inner canine with two apical teeth, including a larger inner one with a serrated inner margin near the apex with three spines. Three long serrated bristles arising from the base of the inner canine (Fig. 6G). A single stout feathered seta lateromedially on each mandible (Fig. 6E, 6F). Maxillae (Fig. 6H) crowned by a rigid canine and three subequal moveable dentisetae; three long-feathered stout bristles arising near the base of the apical canine and dentisetae on the galea-lacinia. A single unserrated bristle arising approximately 2/3 of the way down the sclerotised section of the galea-lacinia. Maxillary palpi 3-segmented, segment II the longest, length ratio of maxillary palp segments from basal one to apical: 1.9:2.2:1 (Fig. 6H).
Carapace. Carapace general colouration dark brown, distally with an irregular (zig-zag) yellow pattern at 0.7 times the length of the carapace (Fig. 1D). Two longitudinal ridges on the anterior surface region, and two short ridges on the pale surface area (Figs 1D, 1F, 9A, 9C, 9D). Carapace with one pale-coloured circular depression on lateral side of the anterior region (Figs 1F, 9B) and two pale-coloured striped depressions at the midline. Carapace flange width very narrow. Cuticle of carapace coarsely pitted and interspersed with scale-like structures (Fig. 9E, F). Distal margin of the carapace protruding slightly over the exhalent notch (Figs 1D, 9A). In lateral view, carapace strongly convex, with a convexity (ratio of maximum carapace height to length along the posterior margin of the carapace) range 0.56-0.58. Sternum pitted, with coarse scale-like structures within the triangular sternal plate (Fig. 8D, F).
Legs. Dorsal margin of fore femur with 14 short simple setae (Fig. 7A); ventral margin of fore tibia with nine pectinate setae (Figs 7B, 10C). Anterior and posterior surface of femur covered with scale-like pattern (Figs 7A, 10A). Tarsal claws of all three pairs of legs sharp and without denticles.
Subimago and Imago. Unknown. Etymology. The name carinatum (Latin for carinate or keeled), refers to the prominent keels or ridge-like mesonotal convexity of the species.

Mitochondrial COI sequence analysis
The partial sequences of the mitochondrial COI gene (658 bp) of three species found in Thailand were analysed to investigate species delineation. Ten specimens of Prosopistoma were examined. In this study, we used Baetisca laurentina from GenBank (HM902945 and KR144660) as the outgroup since Baetiscidae is the sister family of Prosopistomatidae (Ogden et al. 2009). The consensus phylogenetic tree of ML analysis is shown in Figure 11. The tree clearly showed a monophyletic clade for the new species (P. carinatum sp. n.). Clade 2 includes P. annamense and P. sinense. The interspecific genetic distance ranged from 31 to 35% among the investigated species (Table 2).  Table 1.

Discussion
The molecular analysis revealed that Prosopistoma carinatum sp. n. is clearly separated from the other species. The molecular analysis suggests the morphological distinction between the three species. Morphologically, the new species differs from the two previously known species (P. annamense and P. sinense) by (i) longitudinal line ridges on the carapace, (ii) a strongly convex carapace, (iii) the sternal plate, (iv) the scale-like structures on the carapace, (v) 7-segmented antennae and (vi) nine pectinate setae on the ventral margin of the fore-tibiae. The larvae of P. annamense and P. sinense were placed in the same clade, and they share the characteristics of 5-segmented antenna and a smooth carapace. Moreover, the sternal plate of P. annamense and P. sinense has smooth surface (Figs 3A, 5A), while in P. carinatum sp. n. it bears distinctive scale-like structures (Fig. 8D-F). SEM examination on the carapace of the new species reveals a covering of circular scale-like structures scattered over the surface (Fig. 9E, 9F). The cuticle patterns of carapace of P. annamense is quite similar to P. sinense (Figs 2A-D, 4A-D), which are smoother than in the new species.
A comparison of the larvae of all Thai species is shown in Table 2. The irregular (zig-zag) pattern which occur on the carapace of P. carinatum sp. n. can also be found in P. annamense and P. leftincki. The comparison between the new species and other known Oriental species indicates close morphological similarity between P. carinatum sp. n. and P. someshwarensis (from India) in terms of the presence of a longitudinal line that looks like a ridge (Roopa et al. 2017). However, the new species can be easily distinguished by its two distinct longitudinal ridges.

Ecology
The beetle-like mayfly larvae Prosopistoma inhabit shallow water under small stones in streams with a moderate to rapid current (Soldán and Braasch 1984, Sartori and Gattolliat 2003, Shi and Tong 2013. It is well documented that Prosopistomatidae are sensitive to pollution (Barber-James 2010). In this study, the habitats of the larvae were generally located in undisturbed upstream sites (forest streams), except P. annamense, which could be found in both disturbed and undisturbed sites. In addition, the larvae of P. annamense were also found in large rivers, including in the large urban river Xiangjiang in China (Yam 2015). In general, the larvae of Prosopistoma are rarely collected. In this study, most of the larvae were found from February through April in streams, with the exception of the southern region, in which they were found in July. Prosopistoma  annamense larvae were found in October in large rivers. The ecological aspects of Thai Prosopistoma, such as microhabitat, feeding, life history and season of larval maturity, should be explored in more detail. In this study, the larvae P. annamense were reared in an earthenware pot with aquatic plants without an extra air supply (Fig. 13). Under these conditions, the larvae can grow to maturity and survive for approximately one month. However, the fully-grown larvae did not moult to the subimaginal stage.
In Thailand, the Isthmus of the Kra region is a widely recognised as a biogeographic boundary (Hughes et al. 2003, de Bruyn et al. 2005, Jantarit et al. 2013. The distribution of the Prosopistomatidae in Thailand is shown in Figure 14. Prosopistoma sinense, which belongs to the 'P. variegatum' clade (Barber-James 2009), is distributed only in the north and west of Thailand, whereas P. annamense is widely spread through northeast, central, and west Thailand. However, neither P. annamense nor P. sinense were found on the other side of the isthmus. The distribution of P. carinatum sp. n. seems to overlap the isthmus region, and this species apparently falls into the 'African' clade. Barber-James (2009) examined the distribution of P. wouterae and suggested that there was a more recent dispersal from the Sunda Islands of this species after the sea level dropped. Prosopistoma wouterae seems to be restricted to the south of the Isthmus of Kra.