A new representative of the genus Bryocyclops Kiefer, 1927 from a karst cave in north-eastern Thailand (Copepoda, Cyclopoida, Cyclopidae) and comments on the generic affinities

The seventh Thai species of Bryocyclops Kiefer, 1927 – Bryocyclops jayabhumi sp. nov. – was found in a karst cave in the Chaiyaphum Province of north-eastern Thailand. The new species differs from all previously-known species by the absence of an inner seta on the proximal endopod of the first four swimming legs. Bryocyclops jayabhumi sp. nov. is most similar to B. maholarnensis Watiroyram, Brancelj & Sanoamuang, 2015 – the monotypic species of Group VII, which was previously described from Thailand. However, the new species differs from B. maholarnensis by having the following characteristics: i) posterior margin of urosomites serrated; ii) anal operculum triangular with acute-tip; iii) P1–P4 Enp-1 without an inner seta; iv) armature on the female P2–P3 Enp-2 and P4 Enp; v) a transformed spine on the male P3 Enp-2. In this study, the generic affinity of the genus Bryocyclops Kiefer, 1927 is discussed and redefined, based on the available literature concerning its principle morphology to fill the present knowledge gap about the characteristics of the genus.


Site description
Chaiyaphum Province sits in the westernmost edge of this plateau, mostly covered by Triassic-Tertiary sedimentary rocks and Permian limestone (Singtuen and Won-In 2018). The Prakai Phet cave of Chaiyaphum Province, located in the Phetchabun Mountain Range, is made of dolomitic limestone karst formed in the Permian Period and it served as a refuge for several Pleistocene mammals (Filoux et al. 2014). This cave is about 1.5 km long, ending in a 45 m-deep abyss and only 15 m of the cave's anterior is open to tourists. The present author visited the cave in the rainy season once a year -September and October 2017-2019 -and a single population of the new copepod on the floor and stalagmites, which are formed by dripping water, was collected at about 4-5 m from the cave's entrance (Fig. 1).

Sampling and specimen preparation
Samples were collected using a hand net (60 µm) from drip pools on the stalagmites and the cave floor and then fixed immediately in ca. 70% ethanol. Adult animals were picked out and preserved with 70% ethanol in 1.5 ml microtube. Adult specimens were dissected under an Olympus SZ51 stereomicroscope in a mixture of glycerol and 70% ethanol (ratio ~ 1:10 v/v). Dissected specimens were mounted in pure glycerol and sealed with transparent nail polish. Permanent slides with dissected animals were examined with an Olympus compound microscope (CX31) at 1000× magnification. Pencil drawings were made with a drawing tube (an Olympus U-Da) mounted on a compound microscope, then the final drawings were scanned for correction in the CORELDRAW 12.0 graphic programme. Specimens for scanning electron microscopy (SEM) were dehydrated in progressive ethanol concentrations (70%, 80%, 90%, 95%, 100% and 100% absolute ethanol) for 15 min each concentration. Specimens were dried in a critical point dryer using liquid carbon dioxide as the exchange medium. Dried specimens were mounted on stubs using adhesive tape under the stereomicroscope. Specimens were coated with gold in a sputter-coater. The SEM photographs were made using a scanning electron microscope (LEO 1450 VP).
The morphological terminology follows Huys and Boxshall (1991). Specimens were deposited at the Natural History Museum, London, United Kingdom (NHMUK) and the Nakhon Phanom University, Faculty of Science, Thailand (NPU).  Type locality. A rimstone pool that is close to an entrance (see site description) in the Prakai Phet Cave (Fig. 1), Thung Luilai Subdistrict, Khon San District, Chaiyaphum Province, north-eastern Thailand; coordinates of cave entrance: 16°29'03"N, 101°47'05"E, altitude: 573 m above sea level. Material examined. Holotype: one adult female dissected and mounted on one slide (NHMUK 2020.48); allotype: one adult male dissected and mounted on one slide (NHMUK 2020.49); paratypes: one adult female dissected and mounted on one slide (NPU 2020-001), three adult females and three adult males preserved in 70% ethanol (NHMUK 2020.50-55), three adult females and three adult males preserved in 70% ethanol (NPU 2020-002). All specimens were collected from the type locality on 9 October 2017 by the author.
Etymology. The species name is taken from the Sanskrit words 'jaya' and 'bhumi', meaning 'land of victory' or 'Chaiyaphum' in Thai, referring to the Chaiyaphum Province, where the new species was collected.
P1-P4 ( Fig. 4A-D). P1-P3 with two-segmented Exp and Enp; P4 with two-segmented Exp and one-segmented Enp. Armature formula (seta in Arabic numerals and spine in Roman numerals from outer-inner or outer-apical-inner margins) as follows:   (Fig. 4A). Intercoxal sclerite with acute distal margins. Coxa without inner seta. Basis with bare, slender outer seta and robust inner spine; setules on inner distal corner. Exp-1 with outer spine. Exp-2 with three spines on outer margin; two apical setae; blunt seta and two normal setae on inner margin. Enp-1 without seta on inner margin. Enp-2 with apical seta and spine; additional seta on outer margin.
Group VII contains two species collected from Thailand -B. jayabhumi sp. nov. and B. maholarnensis Watiroyram, Brancelj & Sanoamuang, 2015. Both species are obviously different from other species due to their P4 Enp, which terminates in two elements instead of five elements (when one-segmented) or four elements (when two-segmented). These species could, however, be differentiated from each other by the following characteristics: i) posterior margin of urosomites serrated (smooth in B. maholarnensis); ii) anal operculum triangular with acute-tip (round in B. maholarnensis); iii) inner setae on medial margin of P1-P4 Enp-1 absent (present in B. maholarnensis); iv) armature on female P2-P3 (each with three elements in the new species, versus four and two elements, respectively, in B. maholarnensis); v) armature on female P4 Enp (two apical elements in the new species versus one apical and one inner seta in B. maholarnensis). The male P3 Enp-2 of the new species has a well-developed, transformed spine similar to those in B. maholarnensis, but with a swollen medial portion.
The new species shows two remarkable characteristics on its swimming legs and in the urosomal serration in both sexes, which have never been seen in other examples of Bryocyclops s. str. The new species lacks inner distal setae on P1-P4 Enp-1 and has a different serrated pattern on urosomites 1-4. The posterior margin on the somites of its congeners have normal serration, with lobes on free margin of hyaline fringe (for example, B. anninae (Menzel, 1926); B. asetus Watiroyram, 2018;B. maewaensis Watiroyram, Brancelj & Sanoamuang, 2012;B. muscicola (Menzel, 1926); B. muscicoloides Watiroyram, 2018;B. trangensis Watiroyram, 2018), versus the new species, which has sparsely indented and a completely smooth hyaline fringe in B. maholarnensis.
Ecology and distribution. The new species has been found in only one locality, about 140 km away from its most similar species (B. maholarnensis). Bryocyclops jayabhumi sp. nov. is so far confined to this locality, while B. maholarnensis has a wide distribution range across Loei and Nong Bua Lam Phu Provinces. Although the salinity of water containing B. jayabhumi sp. nov. was not measured, it is evidently a freshwater species, as its locality is 573 m above sea level and approximately 450 km from the nearest sea. The sampling site, Prakai Phet Cave, is not connected to running water or other groundwater; it is only fed water from the stalactites and tree roots penetrating its ceiling (Fig. 1C). No other copepods were found during the study period.

Discussion
The genus Bryocyclops Kiefer, 1927 is polyphyletic. Variable characteristics determine species in this genus and, as it contains such a complex array of species, it will likely be divided into distinct genera (Dussart and Defaye 2006;Fiers and Van Damme 2017). The diagnostic characteristics for Bryocyclops are incompletely defined because there are deficiencies in the descriptions of most species, especially their antennae, mouthparts, first three swimming legs, integument ornamentations and genital fields. Different authors use different criteria to characterise the genus -except for the male P3 Enp-2, which is uniformly described as having an apical transformed spine, as well as the P4 coxa, which has no inner seta (Rocha and Bjornberg 1987). Kiefer (1927) separated three Java species from the genus Cyclops Müller O.F., 1785: C. anninae Menzel, 1926;C. bogoriensis Menzel, 1926;and C. muscicola Menzel, 1926. From these, he erected the genus Bryocyclops Kiefer, 1927, the species of which were characterised by P5 being completely fused to the thoracic somite, having three setae. Earlier species, described before 1937 and having this reduced P5, were considered members of Bryocyclops.

The original generic affinities
In 1937, Kiefer provided the additional four characteristics of the genus: 1) a genital segment that is wider than it is long, 2) a P4 coxa without inner seta, 3) a male P3 with a transformed spine and 4) an acute distal margin of the intercoxal sclerite of the legs (unspecified legs, but probably P1-P4).
The diagnosis of the genus Bryocyclops s. lato, as well as the status of the groups comprised in it, has been much debated recently and its members have been separated into five genera: Bryocyclops s. str.; Haplocyclops Kiefer, 1952;Palaeocyclops Monchenko, 1972;Rybocyclops Dussart, 1982;and Thalamocyclops Fiers & Van Damme, 2017. Although some authors believe that the Palaeocyclops genus should have remained the subgenus Bryocyclops (Palaeocyclops) Monchenko, 1972, the status of the genus Palaeocyclops is indubitable when considering the possibility of convergences and the morphological differences (Fiers 2002;Dussart and Defaye 2006;Ranga Reddy and Defaye 2008;Fiers and Van Damme 2017).

Remarks on some generic characteristics
Members of Bryocyclops s. str. have acute intercoxal sclerites on their free distal margins, at least on P4. The swimming legs of many early known species (before 1972) are incompletely illustrated and described, except for P4. Regarding the available information, all species of Groups III-VI are characterised by round distal margins in all legs (P1-P4) versus the acute forms on P4 in Groups I, II and VII. In addition, amongst Groups I, II and VII, the anterior legs tend to have rounder distal margins compared to the posterior legs. For example, P1 is round in the females of B. anninae (Menzel, 1926) and B. asetus Watiroyram, 2018(Watiroyram 2018aSanoamuang et al. 2019).
The presence of an inner coxal seta is also, so far, unified for the system of cyclopine genera (Reid and Spooner 1998). This seta and a medial spine at the basis are present on P1, but lacking on other legs in all known Bryocyclops s. str. species and both of these characteristics are identical in the Bryocyclops s. str., Palaeocyclops and Thalamocyclops genera. However, the inner coxal seta is absent on the P1 of some Bryocyclops species, such as B. absalomi Por, 1981, B. asetus, B. maholarnensis and B. jayabhumi sp. nov. Thus, the presence of the inner coxal seta on P1 is a non-significant characteristic for generic diagnosis of Bryocyclops s. str.
All Bryocyclops s. str. species exhibit specialised sexual dimorphism on P3 and P4, which other cyclopids usually express only on the urosomal segmentation, antennules, P5 and P6 (Fiers 2002). Rocha and Bjornberg (1987) note that the transformed spine of the male P3 Enp-2 is traditionally used to determine genus because it is present amongst all Bryocyclops s. str. Additionally, the discovery of new species has confirmed that the apical spine of the male P3 Enp-2 varies in the degree of transformation (its tip, position and ornamentation of medial swelling). For example, B. maewaensis has the greatest medial swelling at the distal half of the spine, produced in the outer margin, similar to most of its congeners; B. jayabhumi sp. nov. has less medial swelling at about one-half of the spine length and is enlarged in both margins; and B. maholarnensis presents the lowest level of transformation. These three species have no ornamentation (spinules) on the surface of their expended parts, but these spinules are present in B. asetus, B. muscicola, B. muscicoloides and B. trangensis. This transformed spine on the male P3 Enp-2 has also been reported in the Thalamocyclops, Palaeocyclops and Siamcyclops genera (Monchenko 1972;Fiers and Van Damme 2017;Boonyanusith et al. 2018). This characteristic has traditionally been used to recognise Bryocyclops and to reasonably conclude that the genera Thalamocyclops, Palaeocyclops and Siamcyclops are more closely related to Bryocyclops s. str. than are Haplocyclops and Rybocyclops. Reid and Spooner (1998) propose that the female P4 Enp of Bryocyclops, which has five elements in most previously-described species (e.g. B. bogoriensis, B. campaneri, B. caroli, B. muscicola, B. muscicoloides, B. trangensis etc.) is considered plesiomorphic. Thus, the presence of two instead of five elements on the P4 Enp in B. maholarnensis and B. jayabhumi sp. nov. (Group VII) is probably apomorphic and new to the genus.
The male P6 of the new species and of all Bryocyclops s. str. (except B. africanus, Group III) and Thalamocyclops species, has three setae. These are considered to be plesiomorphic formations in cyclopines, which have been reduced to two setae in closely-related genera, including Palaeocyclops, Rybocyclops and Haplocyclops. The female P6 of Bryocyclops s. str. and Thalamocyclops are also similar, but they show more reduction than the male forms; the P6 vestiges have three elements in spiniform and setiform, but they always appear in Haplocyclops and Rybocyclops with setae (unknown amongst Palaeocyclops). Amongst seven species collected in Thailand, the P6 of B. trangensis is most similar to Thalamocyclops (but B. trangensis and other Bryocyclops are easily distinguished from Thalamocyclops by the structure of P5), having two setae and one spinule, versus six other species armed with one seta and two spinules (B. jayabhumi sp. nov. with shorter and stronger setae than B. asetus, B. maewaensis, B. maholarnensis, B. muscicola and B. muscicoloides).
The receptaculum seminis is also useful for determining species and genera in the Bryocyclops s. lato. Although this characteristic is still unknown for many precise species, it seems that Bryocyclops species have a developed anterior part of the receptaculum seminis, as do Haplocyclops, Thalamocyclops species: B. anninae; B. caroli Bjornberg, 1985;B. campaneri Rocha & Bjornberg, 1987;and B. jayabhumi sp. nov. (Fiers and Van Damme 2017;Sanoamuang et al. 2019; the present study). On the contrary, both the anterior and posterior portions of this structure are developed in Palaeocyclops and Rybocyclops species (Dussart and Defaye 2001;Ranga Reddy and Defaye 2008). The shape of the spermatophore has, therefore, been used as a generic characteristic to differentiate Siamcyclops from Bryocyclops s. str. Siamcyclops have L-shaped spermatophore structures versus the kidney or bean-shaped spermatophore structures found in most Bryocyclops species (Boonyanusith et al. 2018). However, this characteristic does not fit B. trangensis and B. asetus, which present two other shapes (see Watiroyram 2018a, fig. 3C; Watiroyram 2018b, figs 8C, 9A). It seems, therefore, that the spermatophore structure takes a unique form in Bryocyclops s. str. and, thus, it must be re-observed amongst the earlier discovered species, whose descriptions are lacking this information. The shapes of spermatophore structures vary more amongst cyclopoids than do those in harpacticoids and calanoids, which are generally cylindrical (Williamson and Reid 2009); in this way, spermatophore shapes may be used to determine what species belong to the Bryocyclops s. str. genus.

Conclusion
Bryocyclops jayabhumi sp. nov. is undoubtedly a new species of Bryocyclops s. str. It shares common characteristics with Group VII, showing sexual dimorphism on P3-P4 and the armature of P4. The new species is easily distinguished from its congeners by lacking an inner seta on the Enp-1 of P1-P4. This study asserts that the characteristics of Group VII, sensu Watiroyram et al. (2015) may, therefore, be amended from 'the male P3 Enp-2 without a transformed spine' to 'the male P3 Enp-2 with a transformed spine'. Based on the available information concerning the common characteristics in all described species, a generic emendation of Bryocyclops s. str. is proposed as follows: i) 11-segmented antennule in female; ii) well developed anal operculum; iii) genital-double somite with length/width ratio less than 1; iv) P1-P4 with two-segmented rami or one-segmented rami; v) P1 basis with inner spine; vi) male P3 Enp-2 with an apical transformed spine; vii) P4 intercoxal plate with acute distal margins; viii) P4 coxa without inner seta; ix) spine and setal formula for P1-P4 Exp-2 being 3.3.3.3 and 5.5.5.4, respectively; x) P5 with three elements and without segment; and xi) male P6 with three elements. According to this view, the genus Bryocyclops s. str. contains currently 21 species. Excluded species are as follows: B. constrictus Lindberg, 1947;B. travancoricus Lindberg, 1947;and B. africanus Kiefer, 1932.