Tardigrades of the Ivohibory forest (south-central Madagascar) with a description of a new Bryodelphax species

Abstract In this paper, samples of mosses and lichens collected from Ivohibory Forest (south-central Madagascar) were studied in search for tardigrades. In total, 39 tardigrade taxa were identified, 21 of which are new records for Madagascar. Moreover, one of these is a new species for science. Bryodelphax beniowskii sp. nov., described here based on morphological analysis. It is most similar to Bry. olszanowskii and Bry. parvuspolaris, but differs from them mainly by ventral plates arrangement and some other morphometric characters.


Introduction
Madagascar is the world's fourth-largest Island located in the Indian Ocean around 400 km to the east of the eastern shores of Africa. It stretches between ~12° to ~26°S latitude, occupying in total ca. 590,000 km 2 . Although originally a part of Gondwana, Madagascar Plate broke away from it together with Antarctic, Indian and Australian plates. Madagascar finally separated from the Seychelles and India ca. 66-90 million years ago, leading to its current form. Due to this unique history, the Island is sometimes called a microcontinent (de Wit 2003;Kusky et al. 2007).
Madagascar is characterised by a tropical climate with two climatic zones, each with its own distinct type of vegetation formations. The eastern side of the Island is covered in various evergreens, while its western part is mostly covered by deciduous flora. These zones are divided by a central mountain range, stretching along north-south axis of the Island (Du Puy & Moat 1996). Both zones are divided into several regions, each of which has distinctive climatic characteristics and a set of unique habitats.
Madagascar is characterised by high biological endemism, estimated at > 90% for terrestrial vertebrates and > 80% for vascular plants, and many of the taxa recorded on the Island are only found in one or very few localities (Goodman & Benstead 2003;Wilmé et al. 2006;Callmander et al. 2011). Currently, there are few taxa that are believed to be leftovers from the time when Madagascar was part of Gondwana, while most are assumed to be results of influx from Africa and Asia and subsequent speciation (Yoder & Nowak 2006;Warren et al. 2010;Buerki et al. 2013).
In the present paper, 122 samples collected in Ivohibory Forest area were studied and 39 tardigrade taxa are reported, seven of which are taxa not recorded outside of Madagascar. Furthermore, a new species for science of the genus Bryodelphax Thulin, 1928 is described based on morphological characters.

Materials and methods
Samples and sample processing: Samples of lichens and mosses were collected in the Ivohibory Forest in June 2017 (permits No 122/17/MEEF/SG/ DGF/DSAP/SCB.Re and 150NEV06/MG17). Later they were packed in paper envelopes, dried at a temperature of ca. 30°C and sent to the laboratory at the Faculty of Biology, Adam Mickiewicz University in Poznań, Poland. There, tardigrades and their eggs were extracted from the samples and studied following the protocol of Dastych 1980b. Microscopy and imaging: Adult specimens and eggs for light microscopy were mounted on microscope slides in Hoyer's medium and secured with a coverslip and then examined under an Olympus BX41 phase contrast light microscope (PCM) with an Olympus SC50 digital camera (Olympus Corporation, Shinjuku-ku, Japan).
All figures were assembled in Inkscape 1.0.1. For deep structures that could not be fully focused on a single photograph, a series of 2-10 images were taken every ca. 0.5 μm and then manually assembled into a single deep-focus image in Adobe Photoshop 21.0.2.
Morphometrics and morphological nomenclature: Measurements of all morphological traits are given in micrometers [μm]. Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. The lengths of the claw branches were measured from the base of the claw to the top of the branch. The sp index is the ratio of the length of a given structure to the length of the scapular plate (scp) expressed as a percentage (Dastych 1999). It was later proposed as the psc index by Fontoura and Morais (2011). The ventral plate configuration in Bryodelphax species is given according to Kaczmarek et al. (2012). The genus abbreviations follow Perry et al. (2019).

Results
In total, 1873 specimens and 314 eggs were found in 73 samples. They were identified as belonging to 39 different taxa (see Table I, Table II

Material examined
Five specimens: holotype (female) and four paratypes (all females) mounted on microscope slides in Hoyer's medium.
Description of the females (for measurements and statistics, see Table II): Body yellow or light orange in living specimens (transparent after mounting on microscope slides in Hoyer's medium) ( Figure 1A, B). Eyes absent or not visible after mounting. Small and conical primary and secondary clavae. Cirri interni and externi with poorly developed cirrophores. Cirri interni always shorter than cirri externi. Only lateral cirri A present apart from head appendages. Cirri A of length typical for Bryodelphax, i.e., up to 25% of the total body length.
Dorsal sculpture visible in PCM as black pillars and white pores. Distinctly thicker and darker margins of dorsal plates and internal margins of facets in plates present, as originally reported by Gąsiorek (2018). Pores large and easily detectable and distributed unevenly in scapular plate (6-16 pores/100 μm 2 , x = 10, N = 15 (in total of 5 specimens, three different plate portions for each specimen were measured), Figure 2A) and in caudal (terminal) plate (0-17 pores/100 μm 2 , x = 9, N = 15 (in total of 5 specimens, three different plate portions for each specimen were measured) Figure 2C). In other plates, pore density much lower (0-6 pores/100 μm 2 , x = 3, N = 15 (in total of 5 specimens, three different plate portions for each specimen were measured)). In caudal plate pillars 0.3-0.8 μm in diameter and pseudopores 0.6-0.9 μm in diameter ( Figure 2C). Paired and median plates divided horizontally by smooth stripes into anterior and posterior parts ( Figures 1A, 2B). Median plates 1 and 2 divided by transverse stripes, median plate 3 undivided. Median plate 2 is the largest among all median plates. In addition, paired plates I and II are divided horizontally into two parts. Twelve poorly visible supplementary plates near median plates 1-3 ( Figures 1A, 2B).
Ventral side with eight rows of faint greyish plates (formula VIII:2-2-4-2-4-2-4-1) (Figures 3, 4). First row with two plates situated at the level of head plate, i.e. in front of legs I, second row with two plates and third row with four plates situated between legs I and II, fourth row with two plates and fifth row with four plates situated between legs II and III, sixth row with two plates situated in line of legs III, seventh row with four plates situated in line with the gonophore and eighth row with one plate situated behind the gonophore. Three triangular genital plates (parts of rows seventh and eighth) surrounding the gonopore (two lateral, in line with the gonopore and the third one posterior to the gonopore (Figures 3, 5C). Pillars (0.3-0.5 μm) and pores (0.4-0.5 μm) on ventral plates visible under PCM.
Papilla-like structure on leg I hardly visible under PCM ( Figure 5A), papillae on leg IV present ( Figure 5C). External claws smooth, but internal ones with spurs facing downward and positioned close to the claw bases ( Figure 5B). Female gonopore with the typical six-petal rosette ( Figures 1B, 5C). Males, juveniles and eggs not found.

Remarks
Presence of pores on ventral plates ( Figure 3) makes this species the first one in the genus Bryodelphax to have this distinct morphological characteristic.   Cuénot, 1929 has the type locality in France and it is known mainly from European localities, but is recorded also from Africa, Asia and North America (McInnes 1994;Mcinnes et al. 2017). Some possible taxonomic confusion regarding this species was discussed by Zawierucha et al. (2014). In our study, only one damaged specimen was found, making the final identification impossible. 14. Hys. dujardini (Doyère, 1840)* 6(1), 139 (1), 152(1), 191(1), 199 (8), 241 (1), 329(1) Hypsibius. dujardini, has been reported globally (e.g. McInnes 1994, McInnes et al. 2017. Morphological characteristics of our Madagascan specimens fit well with the recent redescription of this species ). 15. Kri. tessellatus (Murray, 1910 (7), 319 (1) The accurate identification of the species was not possible due to lack of eggs, especially as two other species of the hufelandi group were found in the present study.  (2) The accurate identification of the species was not possible due to lack of eggs which are crucial for correct identification of Mesobiotus species (Kaczmarek et al. 2018c(Kaczmarek et al. , 2020a. However, it is highly probable that these specimens belong to Meb. fiedleri.  (9) Our specimens perfectly agree with original description (Roszkowska et al. 2016). It is reported for the first time outside of its type locality in Argentina. 30. Mur. cf. hastatus* 148(5 + 9), 151(5 + 4) Type locality in Scotland. This relatively rare species has a large Palaearctic distribution (Mcinnes et al. 2017). Although our specimens agree with original description (Murray 1907b;Ramazzotti & Maucci 1983 with English translation by Beasley 1995), we reason that this species needs redescription due to incomplete diagnosis. 31. Neb. cirinoi (Binda & Pilato, 1993)

Discussion
In recent years, the number of known tardigrade taxa from Madagascar almost doubled from 13 in  (1) Adult specimens morphologically similar to Ramazzottius oberhaeuseri (Doyère, 1840). However, the final identification of the species was not possible due to the lack of eggs. 39. Urs. cf. cameruni* 34(1) Despite fitting the description of the species (Iharos 1969), accurate identification of the species was not possible due to presence of a single specimen.
2003 to 23 at the time of the current study (Maucci 1993;Pilato & Lisi 2003;Bartylak et al. 2019;Kaczmarek et al. 2019;Gąsiorek & Vončina 2019;Gąsiorek et al. 2019a;Grobys et al. 2020;Kaczmarek et al. 2020a;Roszkowska et al. 2020;Kuzdrowska et al. 2021). In this study we present further 21 taxa (including one new for science) reported from Madagascar. However, the taxonomic status and distribution of some of newly reported species need to be clarified. Bryodelphax beniowskii sp. nov. is the second species of the genus Bryodelphax reported from Madagascar, but it should be mentioned that Bry. parvulus Thulin, 1928 was reported from the Island long time ago (Maucci 1993). Taking into consideration that the type locality of Bry. parvulus is in Lapland (Thulin 1928) and since that time many new species of this genus were described (Degma et al. 2021), its presence in Madagascar needs confirmation.
Three species found in present study were previously known only from Africa, i.e., Diphascon zaniewi Kaczmarek & Michalczyk, 2004, Minibiotus africanus Binda & Pilato, 1995and Ramazzottius szeptycki (Dastych, 1980a. These species should be temporarily considered as species restricted to Africa. Furthermore, three species, i.e., Mil. argentinum Roszkowska, Ostrowska & Kaczmarek, 2015, Min. pseudostellarus Roszkowska, Stec, Ciobanu & Kaczmarek, 2016 and Doryphoribius amazzonicus Lisi, 2011 were up to now reported only from South America and their presence in Madagascar might suggest a long distance, anthropogenic dispersal. A similar distribution was recently presented for Minibiotus pentannulatus Londoño, Daza, Lisi & Quiroga, 2017 which was described from South America and later reported in South Africa by Stec et al. 2020a). Similarly, Pam. savai (Binda & Pilato, 2001) reported here were recorded from distant locality in Sri Lanka. Considering these discoveries, these species can be potentially considered longdistance migrants.  Bartylak et al. Echiniscus cavagnaroi Schuster &Grigarick, 1966 andKri. tessellatus (Murray, 1910) are known from tropical or subtropical regions and should be considered as likely pantropical species. Moreover, it should also be mentioned that Kri. walteri (Pilato & Lisi, 2003) (Gąsiorek et al. 2021a). The next member of the genus Echiniscus, i.e., Ech. testudo (Doyère, 1840) is considered as species with wide distribution. Reports of this species from localities in Africa, South and North America or New Zealand may suggest a cosmopolitan distribution of this taxon, probably associated with anthropogenic dispersal (Jørgensen et al. 2007;Gąsiorek et al. 2019d).
Specimens of Hys. dujardini found in the present study fit recent redescription of this species ). Its distribution is now very unclear. This species was considered cosmopolitan in the past (McInnes 1994), but almost all records of this taxon need a confirmation based on modern taxonomy.
Two taxa were identified as Mur. cf. hastatus and Hys. cf. convergens. These species have a very unclear taxonomic position and a redescription of both taxa, from the type localities, is necessary. Both species were described from Europe (Murray 1907a;Urbanowicz 1925) and it is highly probable that Madagascan specimens should be considered as different species, however this confirmation will only be possible after     (Iharos, 1969) and Mac. drakensbergi Dastych, 1993 on Madagascar is highly probable as both species were described from Africa (Mcinnes et al. 2017).
We also identified many specimens only to the genus level or to the group of species i.e. Ramazzottius sp., Doryphoribius sp., Mac. hufelandi group, Mesobiotus sp., Pam. richtersi group and Pseudechiniscus (Meridioniscus) sp. Correct identification of these individuals was not possible due to lack of eggs or small number of specimens. It should also be stated that some of the specimens of Mac. hufelandi group, Mesobiotus sp. or Pam. richtersi group may belong to species reported from Madagascar in previous studies or in the present study, however, without eggs, it is not possible to say so with certainty.
In summary, tardigrade fauna of Madagascar is very poorly known but seems to be very rich because even in a very small area studied in the present research, many new species for science were found Bartylak et al. 2019;Roszkowska et al. 2020;Kaczmarek et al. 2020a, b;Kuzdrowska et al. 2021, as well as the present study), altogether with many species new for Madagascar.

Disclosure statement
No potential conflict of interest was reported by the author(s).