Osteology of the posterior vertebral column and caudal skeleton of marine amphibious gobies (mudskippers) (Teleostei: Gobioidei)

Mudskippers are amphibious gobies (Teleostei: Gobioidei, Oxudercinae) that have served as models for the specialised physiology and behaviour of fishes out of water. In this study, a comparative analysis of the posterior vertebral column and the caudal skeleton of ten mudskipper species was conducted on the basis of X-ray imaging. The species considered were Apocryptes bato , Apocryptodon madurensis, Boleophthalmus dussumieri , Oxuderces dentatus , Periophthalmodon freycineti, Pn. schlosseri, Periophthalmus novemradiatus, Ps. waltoni , Pseudapocryptes borneensis , and Scartelaos tenuis . For the osteological description the new term 'modified caudal vertebra' is used for all those vertebrae that display visibly modified neural and/or haemal spines compared to the spines of a 'usual' caudal vertebra, but are not involved in the support of caudal rays. The results reveal that the most terrestrial forms ( Pn. freycineti, Pn. schlosseri, Ps. novemradiatus, Ps. waltoni ) possess distinct traits that are seldom found in the other species. Among these features are (a) the existence of at least two modified caudal vertebrae (also present in S. tenuis ), (b) a particularly close, dovetailing association between the neural spines of the preural vertebrae two and three (restricted to Ps. novemradiatus and Ps. waltoni ), and (c) thickening and shortening of the ventralmost principal caudal rays (also present in B . dussumieri and S. tenuis ). These findings support the idea that the posterior caudal vertebrae and caudal skeleton of the mentioned species are modified to enhance locomotion on land. Moreover, a relationship between character development and degree of terrestrial adaptation is probable, as all three traits are most pronounced in Ps. waltoni , which correlates with its strikingly high level of adaptation to amphibious life. A further aspect of this study is that the newly recognized skeletal structures have good fossilization potential and could therefore facilitate recognition of fossil species of mudskippers, which are currently unknown.


| INTRODUC TI ON
Mudskippers are small to moderately sized amphibious fishes that belong to the Gobiidae sensu Gill & Mooi, 2012 and constitute the subfamily Oxudercinae (Murdy, 1989;. Mudskippers are widely distributed in coastal mangrove and mudflat areas in the tropics, with the exception of the New World (Hoese, 1984;Murdy, 1989;Parenti & Jaafar, 2017). They are characterized by specialised behaviours and the ability to use their limb-like pectoral fins for locomotion on land (Harris, 1960;Sayer, 2005 Murdy, 1989 (one species) (see . Of these, only members of four genera spend time on land as part of their daily life cycle, namely Boleophthalmus, Periophthalmodon, Periophthalmus and Scartelaos (Murdy, 1989).
The objective of this study is to gain a better knowledge of the posterior vertebral column and the caudal skeleton of mudskippers.
These structures are of central importance for locomotion in aquatic vertebrates (Lauder, 1989). For mudskippers, the caudal skeleton is also important for locomotion on land (Harris, 1960;Pace, 2017;Swanson & Gibb, 2004). The specific aim was to examine if certain traits can be detected that may be associated to adaptations to terrestrial locomotion.
Species identification was verified in each case based on the speciesdiagnostic characters, the sampling location and further information provided in Murdy (1989). A specimen of Gobius niger Linnaeus,   (Figure 1b). The usual condition in gobioids is that PU2 possesses an expanded haemal spine (which supports caudal rays, see Figure 1b), but a shortened neural spine (see Fujita, 1990). Also the neural and/or haemal spine of PU3 are visibly differentiated from a 'usual' caudal vertebra -i.e. more elongated, or more expanded, or more bent -and the distal K E Y W O R D S amphibious goby, Gobiidae, osteology, terrestrial adaptation tips of the PU3-spines are associated with (or near to) the caudal cartilage that supports the dorsal and ventral procurrent caudal rays (see Fujita, 1990). Further elements of the caudal skeleton of gobiids are two large hypural plates (Hy1+2, Hy3+4), an autogenous small hypural plate (Hy5), a parhypural (Php) and one or two epural bones (Ep1, 2) ( Figure 1b). The hypural plates and TA B L E 1 List of studied species and locality data. N, number of specimens the parhypural support the principal caudal rays and the epural bones are associated with the support of the dorsal procurrent caudal rays (Figure 1b).

| Study of osteology
For the purpose of this study, we use as additional term 'modified caudal vertebra' (MC), which is numbered from the posteriormost to the anteriormost (like the preural vertebrae). This term is used for each vertebra that has, like a preural vertebra, a visible modification of its neural and/or haemal spine in comparison to a 'usual' caudal vertebra, but differs from a preural vertebra as it is not involved in caudal ray support (Figure 1b). The same terminology and definition are used in Charmpila et al. (in press).

| RE SULTS
Representative X-ray images of each species, with depictions of their preural and modified caudal vertebrae, are provided in the Appendix Figures A1 and A2.

| The posteriormost vertebral column
The presence of two preural vertebrae (PU2, PU3), as seen in Gobius niger (Figure 2a), and the absence of modified caudal vertebrae in F I G U R E 2 X-ray images showing the posteriormost vertebral column and the caudal skeleton of a typical gobiid (a) and of the mudskipper species Apocryptes bato (b), Oxuderces dentatus (c), Apocryptodon madurensis ( Table 1; NMP, National Museum Prague. Scale bars = 5 mm the definition of the present study can be considered as the usual configuration in the Gobiidae; it also occurs in As. bato, O. dentatus, and An. madurensis (Figure 2b-d, Appendix: Figure A1).
Most noticeable is a particular close association between the neural spines of PU2 and PU3 in Ps. novemradiatus (Figure 3d) and Ps. waltoni (Figures 3e, 4i): the PU2 spine has a convex anterior margin that dovetails with the concave posterior margin of the PU3 spine.  Table 1. Scale bars =5 mm

| Caudal skeleton
The configuration of the hypural plates is largely the same in all ten species (Figures 2, 3, Appendix: Figures A1 and A2). There are two large hypural plates (Hy1+2, Hy3+4), approximately equal in size and sepa- This is most pronounced in Ps. waltoni, and least developed in S. tenuis.

| D ISCUSS I ON
Several of the studied mudskipper species displayed at least one modified caudal vertebra (versus not present in other gobiids), with highest numbers in Pn. schlosseri, Ps. novemradiatus, and Ps. waltoni (Table 2).
Modified caudal vertebrae may provide extra robustness to the posterior vertebral column, and thus help support and stabilize the body when the fish moves on land. This would be in line with the occurrence of the highest counts of modified caudal vertebrae in Pn. schlosseri, Ps. novemradiatus, and Ps. waltoni, as these species are among the most TA B L E 2 Summary of the distinctive traits of the posteriormost vertebral column, caudal fin skeleton and caudal rays of the studied mudskippers; species are arranged (from left to right) according to the increase in specific characters
The dovetailing configuration of the neural spines of PU2 and PU3 seen in the two studied species of Periophthalmus (Figure 4h, i) is also visible in the drawings of the caudal skeleton of Ps. kalolo by Fujita (1990: fig. 499) and Ps. barbarus by Harris (1960: fig. 4). Harris (1960) explained it as an adaptation that enables the fish to execute powerful terrestrial 'jumps' ('skipping' according to Harris, 1960, because the animal uses its tail to propel its body into the air, rather than the hind limbs, as in true jumping). Accordingly, the very close association between the neural spines of PU2 and PU3 represents a strengthening of these vertebrae, to withstand the strong forces that act upon them during skipping. That it is solely present in species of Periophthalmus correlates well with their high levels of adaptation to amphibious life (see Polgar et al., 2017).
A thickening and shortening of the ventralmost principal caudal rays was previously noted only for Periophthalmus (Harris, 1960;Murdy, 1989 (Pace, 2017;Zander, 2011), and the strengthening of the ventralmost principal caudal rays is an adaptation that supports these modes of locomotion (Harris, 1960

DATA AVA I L A B I L I T Y S TAT E M E N T
X-ray images of all studied specimens are available upon request to the corresponding author.