A Functional-Morphological Study on the Attachment, Respiration and Feeding Mechanisms in Balitorinae (Balitoridae, Teleostei)

Faculty of Sciences Department of Biology

Research group: Evolutionary Morphology of Vertebrates

Academic year 2012-2013

A functional-morphological study on the attachment, respiration and feeding mechanisms in Balitorinae (Balitoridae, Teleostei)

De Meyer Jens

Supervisor: Dr. Tom Geerinckx Thesis submitted to obtain the degree of Tutor: Dr. Tom Geerinckx Master in Biology

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Index

1. Introduction 1 2. Materials and Methods 6 2.1 Specimens 6 2.2 Kinematics 7 A. Recordings 7 B. Digitization and analysis 8 2.3 Morphology 9 A. Clearing and staining 9 B. Serial sections 9 C. Computed tomography 10 3. Results 11 3.1 Osteology 11 A. Osteology of Beaufortia leveretti 11 Neurocranium 11 Ethmoid region and infraorbital series 14 Hyopalatine arch and opercular series 15 Lower jaw 16 Hyoid arch 17 Pectoral girdle 18 B. Osteology of Sewellia lineolata and Pseudogastromyzon myersi 19 C. Osteology of Botia marcracantha, B. loachata and Lefua costata 21 3.2 Myology 22 3.3 Kinematics 29 A.1 Respiration - timing 29 A.2 Respiration - movement 34 B.1 Feeding - timing 37 B.2 Feeding - movement 38 4. Discussion 40 4.1 Osteological variation 40 4.2 Feeding and respiration 44 4.3 Attachment 49 4.4 Movement 54 5. Conclusion 55 6. Acknowledgements 57 7. Summary – Samenvatting 58 7.1 Summary 58 7.2 Samenvatting 61 8. References 64

1. Introduction

Functional morphology studies the morphology of structures such as muscles and bones and relates this to their function. To understand how morphology is linked to their functioning, it is useful to investigate specialized species. Specialization has no strict definition and is used in several articles with a different meaning. The existing definitions can be placed in three main categories, depending on their point of view: ecological, mechanistic and evolutionary specialization, respectively (Ferry-Graham et al. 2002). These definitions have in common that a specialist can obtain certain resources (food, shelter, …) more easily than a generalist, which uses a wide range of resources.

Specialization is most easily observed in species that have well pronounced or extreme structures. As stated by Adriaens & Herrel (2009), many insights of biological problems can be obtained by studying extreme morphologies. These studies can not only provide us information about how a species adapts to its specific niche, but also allow us to get a better understanding of how natural selection works. Selection mostly favors average phenotypes in most environments, because these can be highly variable (Ridley 2003). However, both disruptive and directional selection allows well pronounced morphologies to develop in the range of all possible phenotypes (Santos 1996, Benkman 2003, Bolnick 2004). These morphologies are thus only selected when they cause a fitness benefit in their ecological environment (Ridley 2003). Studies focused on extreme specializations range from the extreme length of viper fangs (Cundall 2009), over the long ballistic tongue of the Hydromantes (Plethodonthidae) (Deban & Wake 1997), to the specializations of the snout of seahorses (Roos et al. 2009, Leysen et al. 2011).

Teleost fishes are the most species-rich and morphologically diverse group of vertebrates (Nelson 2006, Wainwright 2006, Mehta 2009). Because of this diversity, teleosts have been used in many morphological and ecological studies, especially in studies related to feeding (e.g., Lauder 1983, Clifton & Motta 1998, Grubich 2003, Waltzek & Wainwright 2003, Janovetz 2005).

The tropical Asian rivers contain very rich and diverse fish communities (Lowe-McConnell 1987, Dudgeon 1999, 2000), but ecological research on these species, especially those of low commercial value, is most often neglected (Jacob & Suryanarayanan 1990, de Silva 1991, Roberts 1993, Martin-Smith 1998a, Dudgeon 2000). Fishes inhabiting fast-flowing rivers need specialized modifications in their morphology to prevent themselves from being swept away by the strong water currents. These modifications are mostly related to the formation of adhesive structures at the anterior end of the ventral body side and on the fins. Hora (1922, 1930) already studied a variety of torrential fishes in function of their adaptations to life in fast currents and rocky substrates. He superficially described the attachment capabilities of Balitoridae, formerly known as Homalopteridae (Hora 1932, Kottelat 1988, ICZN 1993). Balitoridae, commonly called river or hill stream loaches, are a diverse group of around 600 species, divided over 59 genera (Nelson 2006). They are native to Eurasia and Africa, with the highest diversity in Asia (Nelson 2006) and still new species are being described (Gu & Zhang 2012, Lokeshwor & Vishwanath 2012, Yang et al. 2012). A fair number of these 600 species are nowadays found in aquarium trade.

Balitoridae are subdivided in two subfamilies, Balitorinae and Nemacheilinae (Sawada 1982, Nelson 2006; Fig 3). The monophyly of the family, however, has been questioned (Slechtova et al. 2007), and some researchers prefer the separate families Nemacheilidae and Balitoridae (Conway 2011, Staab et al. 2012). As stated before, there is almost no information about the ecology of Balitoridae sensu lato (Dudgeon 2000), because most studies are related to taxonomy, phylogeny or external morphology (Hora 1930, Kottelat 1988, Roberts 1993, Tang et al. 2006, 2011). Most Balitoridae are primarily herbivorous, feeding on algae, but they are also dependent on animal food, such as benthic macro-invertebrates and aquatic insects (Mantel et al. 2004, Ward-Campbell et al. 2005, Herder & Freyhof 2006, Beamish et al. 2008). Martin-Smith (1998a) found that in Malaysia the riffles in rivers are dominated by Balitoridae. Riffles are associated with high velocity and turbulence and to live in such circumstances, Balitoridae developed certain specializations.

This study focuses on the subfamily Balitorinae, which is characterized by an anteriorly flattened body and restricted gill openings, placed above the pectoral fins. They have a permanent ventrally oriented mouth. To prevent being washed away by the fast currents, they have developed a form of sucking ability. The pectoral and pelvic fins are inserted horizontally and have been expanded so they can be used as sucker-like adhesive organs (Yang & Dudgeon 2009). These fins allow them to cling to rocks (Banister et al. 1998).

Figure 1: Left: Distribution of the investigated species. Right: Pictures of their natural habitat1. For this research, four species were selected that are characterized by pectoral fins overlapping the pelvic fins and by strongly reduced to absent barbels. Sewellia lineolata and Beaufortia leveretti exhibit a more dorsoventrally flattened body, whereas the body of Pseudogastromyzon myersi and Gastromyzon punctulatus (Fig 2) is less flattened. These fishes are found in fast-flowing rivers in different areas of Asia (Fig 1). By studying these fishes, a more detailed description can be made about the specific attachment mechanisms. By comparing these mechanisms with those of other species using attachment, it is possible to determine if there are multiple solutions for suction behavior or if convergent evolution has taken place. This study also gives attention to the feeding and respiration structures and mechanisms in Balitorinae to find out if there are trade-offs between suction behavior on the one hand and feeding and breathing on the other hand.

1 Source: www.loaches.com/articles/sewellia-lineolata-natural-habitat-and-how-they-get-to-our-aquariums

Therefore the main aims are:

1. Describing the internal morphology, with the aim of identifying the anatomical structures and muscles that are used during attachment, respiration and feeding. More than one species is being studied to detect any structural variation present between species.

2. Understanding how structures such as fins and head parts are acting during feeding and non-feeding stages and how they are involved in attachment in still and flowing water by a kinematic study. Several species are used to make an estimation of possible kinematic variation among those species.

3. Making a comparison of the morphology with specimens and descriptions of related taxa (subfamily Botiinae (Cobitidae), subfamily Nemacheilinae (Balitoridae)) to identify possible autapomorphies (Table 1) and a comparison with species of other orders (Siluriformes, Perciformes) and cypriniform families (Cyprinidae, Catostomidae, Gyrinocheilidae and Psilorynchidae) using attachment to find out how other species prevent being swept away.

Figure 2: The investigated species. A: Beaufortia leveretti. B: Gastromyzon punctulatus2. C: Pseudogastromyzon myersi. D: Sewellia lineolata.

2 Source: http://honffygabor.uw.hu/e107_plugins/halkatalogus/halkatalogus.php?gastromyzon_punctulatus

Next to this, we hypothesize that:

1. Balitorinae are capable of retaining the same respiration rate while being in still or flowing water, as these fishes use their fins for attachment. This would be in contrast to Loricariidae (Siluriformes), which use a suckermouth for attachment (Geerinckx et al. 2007, 2011). 2. We expect respiration rate differences between feeding and non-feeding mechanisms, as there should be a trade-off in the use of the jaw apparatus while feeding or breathing. 3. There are timing differences in the activity of the different structures used for feeding and breathing between feeding and non-feeding conditions. 4. Balitorinae use their pectoral fins to support the removal of water underneath their body to enhance attachment on the substrate.

Figure 3: Phylogeny of the superfamily Cobitoidea. A: Phylogeny based on the study of Sawada (1982). B: Alternative phylogeny modified from the study of Tang et al. (2006)

2. Material and Methods

2.1 Specimens

An overview of the used specimens and their size can be found in Table 1. These species were obtained through commercial aquarium trade, with the exception of Lefua costata. Specimens of these species were loaned from the Florida Museum of Natural History (FMNH 55442). Video recordings of the balitorine species were made to study the kinematics of the species. All animals were anaesthetized in a watery solution of MS 222 (ethyl-3-aminobenzoic acid methanesulfonate salt, Sigma) and were euthanized using a MS222 overdose. These specimens were then fixed using a 4% buffered formalin solution at neutral pH. After washing, the specimens were transferred to 70% ethanol for preservation.

Table 1: summary of the used balitorine specimens. TL = Total length, C & S = Clearing & Staining.

Family- Subfamily3 Species N TL (mm) Use Balitoridae - Balitorinae Beaufortia leveretti 1 57 Recordings, CT-scan 2 64 Recordings 3 72 Recordings 4 61 C & S 5 43 C & S 7 55 Serial section Pseudogastromyzon Balitoridae - Balitorinae myersi 1 35 Recordings, C & S 2 30 Recordings 3 38 Recordings 4 35 Recordings Balitoridae - Balitorinae Sewellia lineolata 1 45 Recordings, C & S 2 39 Recordings Balitoridae - Balitorinae Gastromyzon punctulatus 1 27 Serial section Balitoridae - Nemacheilinae Lefua costata 1 41 C & S Cobitidae - Botiinae Botia loachata 1 36 C & S Cobitidae- Botiinae B. Macracantha 1 49 C & S

3 According to Nelson (2006)

2.2 Kinematics

A. Recordings

For our kinematic study, we made video recordings using a Casio Exilim fh25 high-speed camera. Sequences were filmed at 120 frames per second. We made recordings of Beaufortia leveretti, Sewellia lineolata and Pseudogastromyzon myersi. The individuals were placed in a separate aquarium (19 cm x 9 cm) after which they were allowed some time to adapt to the environment. Recordings were made once before and once after noon to reduce the effect of daytime. Per specimen, at least two recordings of minimal three respiration cycli each were filmed in dorsal, ventral and lateral view while the specimens were exposed to flowing water and while being in still water. So at least 12 recordings were made per specimen (Table 2). Water flow was created by a standard aquarium pump (SicceMicra pump at 400 L/h). Flow speed was determined by digitizing five particles in the water near the specimens and calculating the distance traveled between two consecutive frames (10 frames per particle). The average water flowing speed was 38.72 ± 2.05 cm/s. An example of the arrangement can be seen in figure 4. We were also able to record feeding movement of one individual of each species. For this, we used both feeding tablets4 that were attached to the sides of the aquarium and an individual wall of glass from which algae could be scraped off by the animals. Finally, to study the water flow created by the animals while being attached to the aquarium wall, a solution of milk diluted in water was used. To provide additional information about water flow, recordings were made of fishes moving through turbid water. By following small pieces in this water, we were capable of following the water dynamics around the body.

Figure 4: Aquarium setup for video recordings (specimen in red circle).

4 JBL novo fect and JBL novo tab. Ingredients: molluscs & crustaceans, cereals, vegetables and vegetable by- products, fish and fish by-products, yeast, algae, vegetable protein extracts

B. Digitization and analysis

Per specimen twelve movies were chosen for digitization (Table 2). Points of interest were digitized by using a marker tracking and direct linear transformation (DLT) program (Hedrick 2008) in Matlab (The Mathworks, Natick, MA). Table 2: Number of recordings per specimen used for digitization.

Flowing water Still water Dorsal view 2 2 Lateral view 2 2 Ventral view 2 2

In dorsal and lateral view, these points of interest include the operculum, the tip of the upper snout, the connective tissue between the snout and the pectoral fins and -if possible- the pectoral fins themselves. In ventral view, the upper and lower lips were digitized, together with the pectoral fins and the hyoid arch (Fig 5). After digitization, the x- and y-coordinates were converted to an Excel file and graphs were made. These graphs were filtered using a fourth-order butterworth filter, in order to study the duration of the respiration cycles. Poptools 3.2 (Monte-Carlo Analysis in Excel 2007, 5000 replicates, lower percentile: 0.025; upper percentile: 0.975) was used to test for differences in the duration cycles while the specimen was in still or streaming water. Also a paired t-test, using R gui 1.14.1, was used to check for significant differences between the inspiration and expiration time during breathing vs. feeding.

Figure 5: Used points of interest in Beaufortia leveretti. A: Dorsal view. B: Ventral view. C: Lateral view. CT: Connective tissue, HA: Hyoid arch, LL: Lower lip, OP: Operculum, TUS: Tip of upper snout, UL: Upper Lip. 2.3 Morphology

A. Clearing and staining

From Beaufortia leveretti, Sewellia lineolata and Pseudogastromyzon myersi, at least one specimen was cleared and stained according to the protocol of Hanken & Wassersug (1981). To optimize the results, also KOH (1 to 4%) was used next to trypsin during maceration (Fig 6).

B. Serial sections

Serial histological cross sections were made of one specimen of Gastromyzon punctulatus and Beaufortia leveretti each. These specimens were first fixed in neutralized 10% formalin. After this, the specimens were cleaned with water and dehydrated. Mineralized structures were decalcified. The specimens were then embedded in Technovit 7100. The microtome Leica SM 2500 was used to make sections of 5 µm thick. Staining of the sections was done with toluidine. To study both hard and soft structures with the serial histological sections, a light microscope (Polyvar; Leitz Diaplan) was used. For the cleared specimens, we made use of a Wild M5 and an Olympus SZX-9 stereomicroscope.

Figure 6: Cleared and stained specimens in dorsal view. A: Pseudogastromyzon myersi. B: Sewellia lineolata. C: Beaufortia leveretti (left pectoral fin removed). Scale bar = 1 cm.

C. Computed Tomography

For a specimen of Beaufortia leveretti, a CT scan of the head was made. The scans were performed on HECTOR, a multi-resolution micro-CT system of the Ghent University Centre for X-ray Tomography, developed in collaboration with XRE (X-ray Engineering bvba, www.xre.be). The system consists of an open type directional target X-ray source operated at 120 kV, 233 µA, with a 1 mm aluminium filter. The detector is an aSi flat panel (PerkinElmer XRD 1620 CN3 CS) with a CsI scintillator. For the scan, 2000 projection images of 2000 x 1400 pixels were recorded, covering 360°, with a total exposure time of 1.0 s per projection. Isotropic voxel size was 10 µm. Raw data were processed and reconstructed using the CT software Octopus (Vlassenbroeck et al. 2007). These data were then rendered using VGstudio Max 2.0 (Heidelberg Volume Graphics).

3. Results

3.1 Osteology

A. Osteology of Beaufortia leveretti

In the following part, the osteology of Beaufortia leveretti is given in detail. Terminology follows Conway (2011). The term ‘lacrimal’ is, however, used instead of ‘infraorbital 1’, following Ramaswami (1952).

Neurocranium (Fig 7-8) In dorsal view, the orbitotemporal region exists of the paired frontals, which are broad and flat. Their lateral edge forms the roof of the orbital region. A postepiphysial fontanelle is present posteriorly. This fontanelle is bounded by the large parietals on its lateral side and by the supraoccipital posteriorly. Posterior to the parietal and lateral to the supraoccipital lies the epioccipital, which also lies lateral of the supraoccipital.

In lateral view, the dorsal edge of the autosphenotic is lying to the parietal and the frontals, while its posterior edge contacts the autopterotic, which forms the posterolateral corner of the neurocranium (Fig 8).

Finally, in ventral view, the parasphenoid is observed extending from the ethmoid to the posterior end of the neurocranium. The anterior part is narrow, while the more posterior part starts to broaden just anterior of the prootic, forming a wing-like structure. The posterior end of the parasphenoid bone exists of two slender arms that run towards each other, ending in a point. A ventral extension from the orbitosphenoid towards the dorsal edge of the parasphenoid bone is present. The orbitosphenoid rims the medial surface of the orbit and lies posteriorly to the pterosphenoid, which then rims the posteromedial face of the orbit. The pterospenoid contacts the prootic on its anterior edge with the anterior opening of the trigeminal-facial chamber lying in between. The posterior opening of the trigeminal-facial chamber lies solely in the prootic. The medial edge of this large bone is occluded by the posteroventral wing of the parasphenoid. Its dorsolateral surface contacts the autopterotic, while the posterior region is bounded by the exoccipital on the lateral side and by the basioccipital at the medial side. The basioccipital is the most posterior bone of the cranium, to which the two arms of the parasphenoid are connected.

Figure 7: A: dorsal view of the neurocranium of B. Leveretti. B: Ventral view of the neurocranium, anterior to bottom. Apa: Autopalatine, Apt: Autopterygoid, Asph: Autosphenoid, Boc: Basioccipital, E: Ethmoid, Epo: Epioccipital, Ept: Endopterygoid, Exo: Exoccipital, Fon: Fontanelle, Fr: Frontal, Io: Infraorbital bones, Kin: Kinethmoid, Lac: Lacrimal, Let: Lateral ethmoid, Max: Maxilla, Orsph: Orbitosphenoid, Par: Parietal, Pas: Parasphenoid Pe1: Preethmoid 1, Pe2: Preethmoid 2, Pm: Premaxilla, Pro: Prootic, Ptsph: Pterosphenoid, Se: Supraethmoid, Soc: Supraoccipital, Suo: Supraorbital. Scale bar = 1 mm.

Figure 8: Lateral view of the right side of the neurocranium of B. leveretti, anterior to the right. Ana: Anguloarticular, Apa: Autopalatine, Apt: Autopterygoid, Asph: Autosphenoid, Den: Dentary, E + V: Ethmoid + Vomer, Ecpt: Ectopterygoid, Ept: Endopterygoid, Fr: Frontal, Hm: Hyomandibular, Let: Lateral ethmoid, Max: Maxilla, Mp: Metapterygoid, Op: Operculum, Orsph: Orbitosphenoid, Par: Parietals, Pe1: Preethmoid 1, Pe2: Preethmoid 2, Pop: Preoperculum, Ptsph: Pterosphenoid, Qd: Quadratum, R: Retroarticular, Sym: Symplectic. Scale bar = 1 mm.

Figure 9: Lateral view of the infraorbital series, anterior to the right. Io: 1-5: Infraorbital bones 1 to 5, Lac: Lacrimal. Scale bar = 1 mm.

Ethmoid region and infraorbital series (Fig 7-9) The broad supraehtmoid is firmly attached to the frontals at its posterior edge. It has a short anteromedian prolongation that is more or less rounded and a short expansion at each lateral side. The small prevomer lies ventral to the parasphenoid bones and the ethmoid to which it is fused. It is firmly attached to the first preethmoid on its anterolateral surface. A rather short, cubic, secondary preethmoid, which is round in cross section, is connected by ligaments to the maxilla anteriorly and to the first preethmoid posteriorly. The lateral ethmoid, which separates the orbital and nasal regions, contacts the frontal dorsomedially. Ventrally, it is in contact with the ethmoid, the orbitosphenoid and the first preethmoid. A large lateral process extends ventrally in close contact with the infraorbital bones, ending anteriorly in a groove of the lacrimal. This lacrimal bone is strongly enlarged, extending from the anterior margin of the eye orbit to the tip of the snout, rostral of the premaxilla. A rather broad process is present towards the autopalatine. The first infraorbital bone is long and slender (Fig 9). The third infraorbital is enlarged, exhibiting a large shelf that extends in ventral direction and rims most of the dorsolateral region of the orbit. The other three infraorbital bones exist of ossified sensory canals, with infraorbital splitting up in two, just anterior of infraorbital 3.

The preautopalatine, an endoskeletal bone dorsolateral of the second preethmoid that connects the autopalatine with the maxilla, is very small in B. leveretti. The large autopalatine is extended anteriorly with the most anterior point at the height of the anterior edge of the kinethmoid. A broad anterolateral process that is projecting towards the lacrimal is present, the lacrimal process. A pointed process extends towards the anteromedial process of the ethmoid. The most posterolateral point of the autopalatine articulates with the prominent anterodorsal point of the endopterygoid in a small groove. No overlap with the ectopterygoid is present.

The premaxilla is small and curves towards the midline to its antimere. Each premaxilla has an ascending process that extends dorsocaudally to the kinethmoid, which is short and broad. The kinethmoid, an ossification present between the ethmoid and the upper jaw, has a short anterior process and is relatively large in B. leveretti when compared with related species. It has a complex of ligaments which is attached to the ascending processes of the premaxillae, but also to the neurocranium, the maxillae and the autopalatines (own observation and Staab et al. 2012). The paired maxillae show a pronounced dorsal process and a lateral process for the attachment of the adductor mandibulae. They also exhibit a ventral process towards the ascending process of the premaxilla.

Hyopalatine arch and opercular series (Fig 8) This region consists of the pterygoid bones, the quadrate, the symplectic and hyomandibular, together with the four opercular bones.

The pterygoid region consists of the meta-, endo- and ectopterygoid. The latter has a broad posterior articulation with the quadrate. The metapterygoid is large and curves towards the hyomandibular. At its ventral side, it contacts the symplectic. This symplectic is relatively large and its anterior tip fits in a groove on the medial face of the quadrate. The quadrate itself is large, with anteriorly an articular condyle that fits in the socket of the anguloarticular. It exhibitis a large posterior process extending laterally. The hyomandibular makes contact at its anterior edge with the metapterygoid.

The largest of the opercular bones is the operculum that has a deep groove on its dorsal edge and articulates with the caudoventrally oriented opercular head of the hyomandibular. Anteriorly of this bone lies the preoperculum, which is a rather slender bone that is fused with the posterior process of the quadrate in B. leveretti. The interoperculum is very small and lies medial to the preoperculum. At the ventral side of the operculum, a slender suboperculum is present.

Lower jaw (Fig 8) The lower jaw consists of the typical components: dentary, anguloarticular, retroarticular and Meckel’s cartilage. The dentary has a large coronoid process that extends dorsally. Its posterior region accommodates the anterior portion of the anguloarticular. The anguloarticular has a broad posterior articulation facet for the quadrate (Fig 10), which seems to allow dorsoventral movement only. Its most posteroventral point ends in a tip. A cartilaginous layer, the Meckel’s cartilage, is present on its dorsal surface. The retroarticular is small and conical and articulates with the posteroventral edge of the anguloarticular.

Figure 10: Articulation facet between anguloarticular and quadrate, anterior to the right. Ana: Anguloarticular, Den: Dentary, Qd: Quadrate. Scale bar = 100 µm.

Hyoid arch (Fig 11)

The hyoid arch is mostly similar as those in other fish. A Y-shaped basihyal is present. Ventral to this bone, between the ventral hypohyal tips, lies a paired endoskeletal element, the sublingual. The anterior ceratohyal is longer than the posterior ceratohyal, with its anterior end being broadened to articulate with the hypohyals. Both the interhyal and basibranchial 1 are absent, while basibranchial 2-5 are all present. There are three pairs of hypobranchials and five paired ceratobranchials. Ceratobranchial 5 exhibits large teeth on its main shaft. No upper pharyngeal jaw was present.

Figure 11: Dorsal view of hyoid and branchial arches in Beaufortia leveretti, anterior to top (Dark = cartilage). Bb 2-4: Basibranchiale 2-4, Bh: Basihyale, Cb 1-5: Ceratobranchiale 1-5, ChA: Ceratohyale anterior, ChP: Ceratohyale posterior, Dhh: Dorsal hypohyale, Hb 1-3: Hypobranchiale 1-3, Vhh: Ventral hypoyale. Scale bar = 1 mm.

Pectoral girdle (Fig 12) The cleithrum, a curved bone, is the largest bone of the pectoral girdle, providing a large surface for muscle attachment. It exhibits a groove on its dorsolateral surface, that accommodates the supracleithrum. This elongate bone articulates with the neurocranium on its dorsal edge. Ventromedially, the cleithrum lies in contact with the anterior portion of the large coracoid. The cleithrum is plate-like in ventral view, except at this point of contact. Between the lateral edge of the coracoid and the ventral edge of the cleithrum lies the scapula. On its lateral edge, this bone articulates with the enlarged head of the most anterior fin ray, which is unbranched. Four strongly enlarged pectoral radials are present, supporting the distal radials of 19 branched fin rays. The first pectoral radial lies lateral to the posterior edge of the scapula, while the others articulate with the posterior edge of the coracoid.

Figure 12: Ventral view of the right side of the pelvic girdle of Beaufortia leveretti, anterior to top. Cle: Cleithrum, Cor: Coracoid, DR5: Distal radial 5, PR 1-4: Pectoral radial 1-4, Sca: Scapula. Scale bar = 1 mm.

B. Osteology of Sewellia lineolata and Pseudogastromyzon myersi (Fig 13)

The morphology of the lower jaw, orbitotemporal region and hyopalatine of Sewellia lineolata and Pseudogastromyzon myersi is comparable to the morphology of Beaufortia leveretti. The only difference worth mentioning is the absence of the postepiphysal fontanelle in both S. lineolata and P. myersi. The ethmoid region, however, shows some strong variation. The supraethmoid in S. lineolata is shorter than the supraethmoid of B. leveretti. It lacks the anteromedian prolongation, while the two lateral expansions are elongated. The supraethmoid shape of P. myersi is similar to the one in B. leveretti, although the two lateral expansions are smaller, while the anteromedian prolongation is relatively larger.

The lacrimal is enlarged in all species, but has a different form. While the lacrimal is forming a sharp angle anteriorly in B. leveretti and P. myersi, this angle is absent in S. lineolata. The lacrimals in this latter species run towards each other in a point. The lacrimal of P. myersi has a lateral process that runs towards the infraorbital bones. This process is not found in the other species.

The kinethmoid strongly differs between B. leveretti and S. lineolata. While the kinethmoid of the first is short and broad, it is long and slender in the latter. The kinethmoid of P. myersi is shorter and broader than the one of B. leveretti and contains two grooves at its anterior edge, in which the dorsal process of the paired premaxillae fit.

The autopalatine of S. lineolata is long and slender. A long process that is oriented anteriorly runs towards the maxilla. The lacrimal process is absent. It articulates in a similar way with the endopterygoid at its posterior edge as in B. leveretti. The autopalatine of P. myersi, however, is very broad, covering almost the whole nasal opening. The width of the autopalatine of B. leveretti is therefore intermediate between the width of the autopalatine of S. lineolata and the autopalatine of P. myersi. Next to a broad lacrimal process and a pronounced anteromedial process towards the ethmoid, the autopalatine of P. myersi also exhibits a blunt anterior process towards the maxilla.

Finally, variation is observed in the lateral ethmoid. This bone is broad in B. leveretti. In S. lineolata, however, this bone is very slender. In P. myersi, it is also not as broad and in both P. myersi and S. lineolata, it does not fit in an anterior groove of the lacrimal process. The other structures of the ethmoid region and their connections are strongly similar between all species.

Figure 13: Dorsal view of the anterior region of the neurocranium, anterior to bottom. A: Sewellia lineolata. B: Pseudogastromyzon myersi. Apa: Autopalatine, E: Ethmoid, Ept: Endopterygoid, Fr: Frontal, Io 1-2: Infraorbital bone 1 and 2, Kin: Kinethmoid, Lac: Lacrimal, Let: Lateral ethmoid, Max: Maxilla, Pm: Premaxilla, Se: Supraethmoid. Scale bar = 1 mm.

C. Osteology of Botia macracantha, B. loachata and Lefua costata

Finally, a summation of the most important differences and similarities is made between the investigated balitorine species and cleared and stained specimens of related species (Fig 14). Botia macracantha and Botia loachata are members of Botiinae (Cobitidae), whereas Lefua costata belongs to Nemacheilinae (Balitoridae).

The neurocranium of these three species is comparable with the description of the neurocranium of the investigated Balitorinae, but some differences are found. A fontanelle is present in the observed Botiinae and Nemacheilinae, comparable to the fontanelle found in B. leveretti, but larger. The exoccipitals are separated by the supraoccipital in Balitorinae, but in the other species, the exoccipital lies in direct contact with its antimere.

In all investigated species, the orbitosphenoid lies in direct contact with the ethmoid region. The symplectic, which is broad and exhibits a pentagonal form in Balitorinae (Fig 8), is long and slender in Nemacheilinae and Botiinae. Both the nemacheiline and botiine species lack the large dorsal groove of the operculum that is present in balitorine species.

Most of the differences are, however, found in the ethmoid region of the head. In all investigated species, a second preethmoid is present. The first preethmoid, however, is absent in both Botia species and in L. costata. The autopalatine is not as broad as in balitorine species. Therefore, the preautopalatine is easily visible and it is much larger in comparison with the preautopalatine of Balitorinae. The kinethmoid of L. costata is short and broad, similar to the kinethmoid of P. myersi. This is in contrast to B. macracantha and B. loachata, which have a long and slender kinethmoid, with two pronounced dorsal processes. This kinethmoid morphology is more comparable to the form found in S. lineolata.

The ethmoid bone shows a lot of variation in form among the different balitorine species. This variation is also found in the other species. The supraethmoid has a long and slender form in B. loachata and is small. L. costata exhibits an even smaller supraethmoid in comparison to B. loachata. As a consequence of the small supraethmoid, the strong connection between the ethmoid and the frontals that is present in Balitorinae, is not found in B. loachata and L. costata. This is in contrast to B. macracantha, that exhibits a broad supraethmoid, similar in form to S. lineolata.

The infraorbital bones are all long and slender in the investigated Botiinae and L. costata. This means that the third infraorbital of these species doesn’t have a large extending shelf. Finally, the most pronounced difference between the balitorine species and the other investigated species, is the absence of the enlarged lacrimal. The lacrimal is long and slender in Botiinae and L. costata, whereas it is one of the largest bones in the skull of Balitorinae.

Other structures are similar between all investigated species.

Figure 14: Cleared and stained specimens for comparison. A: Lateral view of Botia macracantha. B: Lateral view of Botia loachata. C: Lateral view of Lefua costata. Scale bar = 1 cm.

3.2 Myology (Fig 15-20)

The myology of Beaufortia leveretti is described in detail, with reference and comparison to Gastromyzon punctulatus when relevant. The names of the muscles are given according to Winterbottom (1974)5.

The adductor mandibulae consists of three major subdivisions. The A1 portion occupies the major region of the cheek in B. leveretti, whereas it is smaller in G. punctulatus (Fig 15-17). It originates on the dorsal edge of the preoperculum and the lateral side of the hyomandibular, with its main insertion on the lateral side of the maxilla by a large tendon. Some fibres of the muscle split off, attaching to the anguloarticular of the lower jaw. More posteriorly, fibres attach to the infraorbital 3. These fibres are not found in G. myersi. No distinction could be made between A1α and A1β (Fig 15).

5 The alphanumeric terminology used for the four main divisions of the adductor mandibulae (A1, A2, A3 and Aω) fails to reflect the homology of these divisions across Teleostei (Diogo & Chardon 2000, Datovo & Vari 2013). An alternative nomenclature scheme was recently proposed by Datovo & Vari (2013).

Figure 15: Lateral view of the head muscles in Beaufortia leveretti based on serial sections, anterior to the left (only most important bones indicated). A1-2: Adductor mandibulae 1-2, AAP: Adductor arcus palatini, AO: Adductor operculi, Ana: Anguloarticular, Apt: Autopterygoid, Asph: Autosphenoid, DO: Dilatator operculi, Fr: Frontals, Hm: Hyomandibular, Io3-4: Infraorbital 3-4, Lac: Lacrimal, LAP: Levator arcus palatini, LO: Levator operculi, Max: Maxilla, Op: Operculum, Pop: Preoperculum, RT: Retractor tentaculi. Scale bar = 1mm.

Figure 16: Lateral view of the head muscles in Beaufortia leveretti based on serial sections anterior to the left (only most important bones indicated). A1 and infraorbital bones removed. A2-3:Adductor mandibulae 2-3, AAP: Adductor arcus palatini, Ana: Anguloarticular, AO: Adductor operculi, Apt: Autopterygoid, Asph: Autosphenoid, Den: Dentary, DO: Dilatator operculi, Fr: Frontal, Hm: Hyomandibular, LAP: Levator arcus palatini, LO: Levator opercula, Max: Maxilla, Mp: Metapterygoid, Op: Operculum, Pop: Preoperculum, Qd: Quadrate, Sym: Symplectic. Scale bar = 1mm.

Medial to the A1 run the bundles A2 and A3 (Fig 16-17). Both muscle bundles are small in comparison with the large A1. The A2 inserts with a small tendon, dorsal on the coronoid process of the dentary in B. leveretti. The insertion of the A2 in G. punctulatus is, however, found on the medial side of the dentary bone (Fig 20B). Origin is on the most posterior point of the metapterygoid. The A3 is the smallest part of the adductor mandibulae. It inserts on the articular, ventral to the A2. At the level of the symplectic, it partially fuses with the A2 (Fig 16 and 20C).

Figure 17: Muscles at the level of the eye in Gastromyzon punctulatus. AAP: Adductor arcus palatini, A1-3: Adductor mandibulae 1-3, PH: Protractor hyoidei. Scale bar = 1 mm. The retractor tentaculi inserts ventrally to the anterior edge of the enlarged lacrimal (Fig 15 and 19A). It originates posteriorly with a large tendon on fat tissue at the ventral side of the body, lateral to the dentary (Fig 19C). The nerve departing from this muscle runs towards the A1.

The adductor arcus palatini is a large muscle, inserting on the metapterygoid and originating on the parasphenoid. The levator arcus palatini originates on the sphenotic and inserts on the hyomandibular. It is a large muscle, but rather short in length and lies ventral of the dilatator operculi (Fig 16 and 20E). This latter muscle is also large and originates on the frontals. It is conical in shape, with its apex lying ventrolaterally. It inserts on the dorsal process of the operculum. The levator operculi inserts on the dorsomedial face of the operculum and originates on the dorsal side of the pterotic. It lies close to the adductor operculi, with a large nerve running between them (Fig 20F). The adductor operculi itself is longer than the levator operculi, originating on the ventral side of the pterotic and inserting on the operculum, just behind its articulation with the hyomandibular (Fig 16). The eye muscles consist of the obliquus inferior, obliquus superior, rectus externus, rectus inferior, rectus superior and rectus internus (not illustrated).

Figure 18: Ventral view of the head muscles in Beaufortia leveretti, anterior to top (only most important bones indicated). BR: Branchiostegal rays, Den: Dentary, HA: Hyoid arch, HI:Hyohyoideus inferior, HAB: Hyohyoideus abductor, IM: Intermandibularis, Lac: Lacrimal, PH: Protractor hyoidei, RT: Retractor tentaculi. SH: Sternohyoideus. Scale bar = 1mm.

Most anteriorly on the ventral surface of the head, the intermandibularis is found. This muscle is very short and small in B. leveretti, but larger in G. punctulatus, and connects both dentary bones (Fig 18). The protractor hyoidei consists anteriorly of two parts, each part attached to a dorsomedial edge of a dentary bone, with the intermandibularis lying in between. More posteriorly, the two parts join and separate again, giving it an X-like shape in ventral view. The muscle originates on the hyoid arch in both B. leveretti and G. punctulatus. The hyohyoideus inferior connects the left and right side of the hyoid arch. Muscle fibres split off of this muscle towards the branchiostegal rays, forming the hyohyoideus abductor. The hyohyoidei adductores, connecting the branchiostegal rays, are not found6. Finally, the sternohyoideus is a large paired muscle that connects the cleithrum of the pectoral girdle and the urohyal bone. It arises dorsal to the posterior end of the hyohyoides inferior.

Figure 19: A-C-E: Retractor tentaculi in Beafortia leveretti. B-D-F: Retractor tentaculi in Gastromyzon punctulatus. A&D: insertion of muscle in B. leveretti B&E: Maximum surface in cross section. C&F: Origin. Io: Infraorbital, Lac: Lacrimal, RT: Retractor tentaculi. Scale bar = 500 µm.

6 This region was damaged in the serial sections, so interpretation was difficult and might need revision.

Figure 20: A-C-D-F: Muscles in Beaufortia leveretti. B-E-G: Muscles in Gastromyzon punctulatus . A-B: Insertion of A2, C: fusion of A2 and A3, D-G: Muscles posterior to eye A1-3: Adductor mandibulae 1-3, AAP: Adductor arcus palatini, AO: Adductor operculi, Apa: Autopalatine, Den: Dentary, DO: Dilatator operculi, E + V: Ethmoid + Vomer, Fr: Frontal, Hm: Hyomandibular, Io: Infraorbital bone, Lac: Lacrimal, LAP: Levator arcus palatine, Let: Lateral ethmoid, LO: Levator operculi, Max: Maxilla, Mp: Metapterygoid, Op: Operculum, Pe1: Preethmoid 1, Pt: Pteryogoid, Qd: Quadrate, Sph: Sphenoid, Sym: Symplectic. Scale bar = 1 mm.

3.3 Kinematics

A.1 Respiration - timing

A lot of variation was found in the mean duration of the respiration cycles of the hyoid arch within and among Beaufortia leveretti, Sewellia lineolata and Pseudogastromyzon myersi (Table 3). Due to this high variation, none of the investigated species showed a significant difference while being in still vs. flowing water (p > 0.05, Monte-Carlo analysis). The cycle duration of the different species is almost the same, with respiration cycles being slightly shorter in flowing water. The variation in the other digitized points was similar to the values in table 3.

Table 3: Cycle duration based on the hyoid movement in the studied species. Beaufortia leveretti: 3 specimens, Sewellia lineolata: 2 specimens, Pseudogastromyzon myersi: 2 specimens. N = number of respiration cycles.

Still water Flowing water P value Mean ± SD (ms) N Mean ± SD (ms) N Beaufortia leveretti Hyoid arch 253.99 ± 47.27 23 225.83 ± 46.19 20 0.261 Sewellia lineolata Hyoid arch 223.25 ± 40.60 19 216.25 ± 36.12 20 0.318 Pseudogastromyzon myersi Hyoid arch 242.26 ± 42.17 14 231.25 ± 28.46 12 0.305

Table 4 shows that the high variation above is not only due to differences between specimens of the same species. High variation can be present in a recording of 3 s of a single specimen (e.g., recording 1 of specimen 2 in flowing water), but also between two recordings under the same conditions of water flow, the variation can be high. For example, during one recording of a Beaufortia leveretti specimen in still water, the respiration time was 352.78 ± 36.11 ms (N=3), while it was 291.67 ± 33.33 ms (N=3) in another recording. From this, it can be derived that high variation can be present at both the individual and species level. The same observations are made in Pseudogastromyzon myersi and Sewellia lineolata. For example, in a recording of a P. myersi specimen being in still water, the respiration duration was 226.67 ± 72.50 ms (N=5). As the condition of water flow did not change in any of these cases, other factors may be responsible for these observations.

Table 4: Mean cycle duration time ± SD based on the hyoid movement in Beaufortia leveretti. Rec 1 = Recording 1, Rec 2 = Recording 2.

Flowing water Still water rec 1 (ms) rec 2 (ms) rec 1 (ms) rec 2 (ms) specimen 1 208.33 ± 16.67 177.08 ± 52.08 225.00 ± 54.17 226.67 ± 39.17 specimen 2 202.08 ± 63.19 208.33 ± 8.33 352.78 ± 36.11 291.67 ± 33.33 specimen 3 255.56 ± 19.44 297.22 ± 19.44 254.16 ± 25.00 216.67 ± 58.33

Figure 21 shows that respiration time is more or less similar when the specimen is out or in flow. The lower lip can be associated with lower jaw movement, while the upper lip movement corresponds to that of the upper jaw. The amplitude differences in the graphs of the hyoid arch are caused by the fact that the hyoid region was not equally well visible in the different recordings, so the exact same points could not be digitized.

Maximal lower jaw depression was reached at the same moment of maximal hyoid depression with a maximum difference of 3.33 ms between the two structures reaching a maximum depression. Also maximal elevation of the lower jaw was reached simultaneously with maximal elevation of the hyoid. This was the case during breathing in both still and flowing water. Inspiration and expiration duration was also measured. During breathing, mean inspiration (108.93 ± 10.57 ms) and expiration time (132.84 ± 14.79 ms) are almost equally long.

Figure 21: Timing of cycling of the different points of interest of the same Beaufortia leveretti specimen in ventral view. Panel 1: Timing in flowing water. Panel 2: Timing in still water.

The time that the operculum was closed and opened was also measured (Table 5). No general trend can be observed among the three species. The operculum of B. leveretti and S. lineolata in flow is longer closed than opened, while the opposite is observed in still water.

In P. myersi, however, the operculum is longer opened in both still and flowing water. Due to the high variation, no significant differences are found between the two conditions of water flow and the species seem to have a similar duration time in still and flowing water (p > 0.05, Monte-Carlo analysis)

Table 5: Overview of the mean time ± SD that the operculum was closed (TC) and opened (TO) for the different species. N: Number of respiration cycles.

Flowing water Still water TC (ms) N TO (ms) N TC (ms) N TO (ms) N B. leveretti 121.21 ± 28.37 22 112.28 ± 34.62 19 115.12 ± 17.91 27 116.67 ± 30.86 22 S. lineolata 103.70 ± 9.62 27 84.67 ± 19.50 26 93.12 ± 11.42 24 99.64 ± 10.80 23 P. myersi 100.98 ± 8.27 17 151.56 ± 16.16 16 90.91 ± 12.61 11 111.36 ± 16.36 11

Finally, pectoral fin movement was registered. To distinguish between still and flowing water, the total number of recorded fin movements and the number of recordings that didn’t show any movement were calculated. In flowing water, a total of 57 pectoral fin displacements were digitized in 37 recordings of B. leveretti specimens, corresponding to a total of 96 respiration cycles (59.38%), whereas the pectoral fin was only moved 30 times during 127 respiration cycles in still water (23.62%). As a similar trend is found in S. lineolata and P. myersi, it is possible to conclude that Balitorinae thus use their fins more when they are exposed to flowing water. However, only 11 of the 37 recordings in flowing water showed movement, while in still water, fin movement is observed in 24 of the 40 recordings. This means that the higher number of displacements in flow is only caused by 29.73% of the recordings, while in the other 70.27% recordings no motion is observed. This is in contrast with specimens in still water, where 60% of the recordings show some pectoral fin motion. The same observations are found in S. lineolata and P. myersi. From this, it can be concluded that the condition of water flow does not have an influence on the frequency of pectoral fin movement.

A.2 Respiration - movement

In ventral view, the upper and lower lip, which correspond to the upper and lower jaw respectively, only move a little and don’t touch each other during respiration. Elevation of the lower jaw increases the volume of a small cavity underneath the attached body which probably results in an underpressure. Elevation of the lower jaw initiates simultaneously with depression of the upper jaw. The skin anterior of the upper lip is pulled posterodorsally, possibly under influence of the retractor tentaculi (see ‘3.2 myology’ and ‘4. discussion’), allowing a small volume of water to flow into the cavity underneath the body. Then, the skin returns anteroventrally and attaches again to the substrate. Next, the lower jaw depresses and the volume of water that entered under the body is taken up through the mouth opening. In both lateral and dorsal view, small dorsoventral excursions of the tissue overlaying the kinethmoid can be observed during breathing.

Water that enters through the mouth is removed through the opercular slit. The pectoral fin, however, can assist in the removal of water underneath the body. The anterior fin rays are covered with unculi -horny projections arising from single cells- on the ventral side, enhancing friction (Fig 22). The most posterior fin rays of the pectoral fin, however, aren’t used for attachment to the substrate and therefore lack unculi. They are attached laterally to the body and are responsible for the removal of water underneath the body. The posterior fin rays detach from the body and create a small gap between the fins and the body through which small volumes of water can escape. After removal of the water, the posterior fin rays reattach to the body (Fig 23).

Figure 22: Cross section of a fin ray in Beaufortia leveretti, with unculi on the ventral side. Scale bar = 100µm.

Figure 23: Fin movement during breathing in Sewellia lineolata. A: Adducted fin rays. B: Start fin ray detachment. C: Maximal fin detachment. The general movement of the studied balitorine species is composed of two steps and is most easily followed in ventral view. We observed that these species prefer to remain attached to the substrate and are only free-swimming when really necessary. They developed a special type of movement to remain attached to the substrate while being able to move. The body of the species (especially B. leveretti) can be described as an hourglass that consists of two parts

(Fig 24A). The anterior part bears the pectoral fins while the posterior part bears the pelvic fins and the caudal fin. These two parts are used as two largely independent suckers while moving. During movement, the anterior part detaches and moves to the left or the right (Fig 24B), whereas the posterior part remains attached. This way, one pectoral fin is brought forward (compare left pectoral fin between 24A and 24B). Next, the anterior part attaches again to the substrate and is used as anchor point, allowing the posterior part to detach and move in the opposite direction as the anterior part (Figure 24C). Balitorine species thus assure that always one part of the body remains attached to the substrate while the other part is free to move. Observations of specimens moving against the current confirm that detachment of the substrate is prevented by the use of the whole body as suckers. Some specimens were even seen moving backwards.

Figure 24: General movement in Beaufortia leveretti. Arrow: Direction of body movement. A: Both suckers attached. B: Movement of the anterior sucker, while the posterior sucker remains attached. C: Movement of the posterior sucker while the anterior sucker remains attached.

B.1 Feeding - timing

During feeding, the time that the operculum was closed and opened was determined. Next to this, the duration of the depression and elevation of the lower jaw was also measured. Maximal elevation and depression of the lower jaw are reached at the same time as maximal elevation and depression of the lower jaw. The elevation of the lower jaw is therefore associated with expiration, whereas depression of the lower jaw is associated with inspiration. Only the data of B. leveretti were taken into account as only three cycles were recorded of the other species, which is not sufficient to be reliable. Feeding was recorded in non-flowing water. The measurement of the duration of operculum movement didn’t show significant differences (Table 6).

Table 6: Mean time ± SD of the time that the operculum was closed (TC), opened (TO) and the total time of one cycle (operculum closed-opened-closed) in Beaufortia leveretti. P-values obtained by Monte-Carlo analysis.

Feeding (1) Breathing p-values Flowing water (2) Still water (3) 1 - 2 1 - 3 N 17 17 11 TC 105.39 ± 20.82 121.21 ± 28.37 115.12 ± 17.91 0.7812 0.9762 TO 95.83 ± 17.83 112.28 ± 34.62 116.67 ± 30.86 1.0000 0.9240 TOTAL 202.78 ± 21.77 235.97 ± 47.10 231.06 ± 37.64 0.9952 0.9910

When a distinction is made between inspiration and expiration time, a significant difference is found between feeding and non-feeding conditions. Whereas inspiration and expiration take an equal amount of time during breathing, this is not the case during feeding (Table 7). The inspiration time during breathing is almost twice as long as during feeding (p < 0.05, paired t- test in R), whereas the expiration time is significantly shorter during breathing (p < 0.05, paired t-test in R). Inspiration during feeding thus happens much faster than during non- feeding. Total respiration time does, however, not differ significantly between feeding and non-feeding (p < 0.05, Monte-Carlo analysis). The lower lip requires 179.17 ± 12.05 ms to close and reopen while feeding. Under non-feeding conditions, this is 222.37 ± 48.52 ms in flowing water and 256.52 ± 45.01 ms in still water, with both showing high variation.

Table 7: Mean inspiration and expiration time during normal breathing and feeding in Beaufortia leveretti.

Inspiration Expiration Breathing Feeding Breathing Feeding N 28 28 14 14 Mean ± SD (ms) 108.93 ± 12.92 59.23 ± 16.88 109.62 ± 12.66 132.84 ± 12.66

B.2 Feeding - movement

As stated above, no significant differences are found in total respiration time between feeding and normal breathing. However, the movement of the skin anterior of the upper lip and the movement of the lower and upper jaw are more pronounced during feeding (Fig 25). Expiration takes more time during feeding and is associated with a stronger elevation of the lower jaw. The lower jaw keeps elevating, even when the mouth is closed. This results in a larger cavity underneath the body, presumably creating a larger underpressure, which leads to an increased suction effect. Simultaneous with this elevation, the skin anterior of the mouth is strongly pulled posterodorsally, allowing a larger volume of water to run underneath the body. The skin moves back anteroventrally and during the short inspiration phase, both water and feeding particles are sucked into the expanding buccal cavity. After maximal opening of the mouth, it is the upper jaw that depresses first and thus initiates closing, whereas the lower jaw remains depressed. After inititiation of mouth closure by the upper jaw, also the lower jaw elevates and the mouth is fully closed. In lateral view, it is observed that the increased elevation of the lower jaw is simultaneous with more pronounced dorsoventral excursions of the skin overlying the kinethmoid.

Figure 25: Feeding movement of Beaufortia leveretti. A: Maximum depression of the lower jaw. B: Elevation of the lower jaw. C: Maximum elevation of the lower jaw.

Similar to during breathing, water underneath the body enters through the mouth and is removed through the opercular slit. However, due to leakage through the gaps between the unculi and the prolonged elevation of the lower jaw, a larger volume of water runs underneath the body. The pectoral fin, therefore, moves more pronounced during feeding. All posterior fin rays become detached from the body simultaneously (Fig 26), creating a large gap between the body and the fins, through which a large volume of the water is removed. This type of fin movement should indeed support water removal, as it is also observed during situations in which the fish is not fully attached to the substrate. Because of this, a larger volume of water slips underneath the body, which is then removed by iterations of this pronounced fin displacement.

Figure 26: Fin movement during feeding in Beaufortia leveretti. A: Normal situation. B: Start detachment. C: Maximal detachment.

4. Discussion

4.1 Osteological variation

The different osteological elements of the neurocranium of Balitorinae were identified and it was observed that the structures of the feeding and respiration apparatus were more similar between Beaufortia leveretti and Pseudogastromyzon myersi. Both these species have following features in common (Fig 7 and 13B):

1. The supraethmoid has an anteromedian prolongation and two lateral expansions. 2. The autopalatine has three processes: a broad lacrimal process, an anteromedial process towards the ethmoid and a blunt process towards the maxilla. The width of the autopalatine of B. leveretti is intermediate between the very broad autopalatine of P. myersi and the slender autopalatine of Sewellia lineolata. 3. The kinethmoid is short and broad, with two grooves where the dorsal process of the maxilla fit in. 4. The anterior portion of the lacrimal is set at a medial angle.

This is not the case in S. lineolata, which differs from the other species in the following features (Fig 13B):

1. The supraethmoid has two large lateral expansions and no anteromedian prolongation is present. 2. The autopalatine is slender, lacking the lacrimal process and the pronounced process towards the ethmoid. 3. The kinethmoid is long and slender. 4. The lacrimal does not form a medial angle.

The observation that the internal anatomy of B. leveretti and the anatomy of P. myersi are more similar to each other than the one of S. lineolata is unexpected when the size of the mouth is taken into account. Both B. leveretti and S. lineolata have a small mouth, while the mouth of P. myersi is broad. Ramaswami (1952) already mentioned that a lot of variation was present in the skull morphology of Balitoridae (then Homalopteridae) and this is also observed in this research. The mouth is not used as the main suction apparatus, but can still play a role in the attachment by removing water from underneath the body. According to the phylogeny of Liu et al. (2012), S. lineolata is the most primitive of these three species, while P. myersi is the most derived (Fig 27). Based on this phylogeny, it can be deduced that there

40 is a trend towards more broad bone structures. Also the medial angle formed by the lacrimal is becoming more pronounced. No conclusions can be made about the possible benefits of this trend towards broader bones.

Figure 27: Phylogeny of Balitorinae according to Liu et al. (2012). The species in this study are used as representatives of Balitorinae with pectoral fins overlapping the pelvic fins. It needs to be noted that not all Balitorinae exhibit such a morphology, as these fins do not overlap in all balitorine species (Fig 29). A comparison between the investigated Balitorinae and other genera was made. Botia macracantha and Botia loachata are members of the related family Botiinae (Cobitidae, Cypriniformes), whereas Lefua costata is a representative of the subfamily Nemacheilinae (Balitoridae, Cypriniformes). The investigated Balitorinae differ with the related genera in following features:

1. The fontanelle, present in the neurocranium, is absent or small in Balitorinae, whereas it is large in Botiinae and Nemacheilinae. 2. The exoccipitals are separated by the supraoccipital in Balitorinae. In the other genera, the exoccipital lies in contact with its antimere. 3. Balitorinae have a broad, short symplectic that has a pentagonal form. This is in contrast to the other genera, which have a long and slender symplectic that is more rectangular in shape.

4. The first preethmoid is present in Balitorinae, but is not found in the other genera. They have a rounded process at the anterolateral corner of the ethmoid- vomer complex instead. 5. The lacrimal bone of Balitorinae is enlarged and extended in anterior direction, whereas this is not the case in Botiinae and Nemacheilinae. 6. The autopalatine is large in relation to the width of the head, combined with a small preautopalatine in Balitorinae. In the other species, the autopalatine is not so large and the preautopalatine is relatively larger in comparison with the one of Balitorinae. 7. Balitorinae have a large supraethmoid which is strongly connected to the frontals. Botiinae and Nemacheilinae have a less developed supraethmoid and the strong connection to the frontals is absent. 8. In Balitorinae, the infraorbital bone 3 is plate-like enlarged (Fig. 9), whereas such an enlargement is absent in the other genera.

The main question is which of the characters of Balitorinae are possible adaptations to torrential life. It is certain that the enlarged lacrimal of Balitorinae is an adaptation to fast- flowing rivers, acting as an immobile anterior border of the sucker body (see ‘4.3 attachment’). This has also been suggested by Ramaswami (1952). The bone is supported by the lateral ethmoid to which it is closely associated. The presence of the plate-like extension of the infraorbital bone 3 is expected to perform a supportive role, as it partially covers the area between the lacrimal and the anterior end of the pectoral fin at the lateral side of the body. The broad and large supraethmoid, with the strong connection to the frontals, could enhance rigidity, which is beneficial to resist the strong current (see ‘4.3 attachment’). The presence of two preethmoid bones and the preautopalatine increases the mobility of the snout (Ramaswami 1952). The first preethmoid bone is absent in the investigated related species, but due to the presence of the rounded process, mobility is retained. The first preethmoid is also present in other Cypriniformes which do not live in torrential rivers (Conway 2011), so these bones are not an adaptation to fast-flowing waters.

We mentioned the high variability in kinethmoid morphology in all investigated species. Both Botia species and S. lineolata have a long and slender kinethmoid, while in the other Balitorinae and L. costata, it is short and broad. Hernandez et al. (2007) already mentioned that these two types are present among the different Cypriniformes and hypothesized that the morphology is associated with feeding behavior, as most benthic species feeding on detritus are associated with long, slender kinethmoids, whereas insectivorous, non-benthic fishes mostly have a short and broad kinethmoid. More recent research by Staab et al. (2012) has shown, however, that kinethmoid shape alone is not responsible for the feeding differences. The three Balitorinae in this research are benthic feeders. They feed primarily on algae, but also insect larvae are included in their diet (Mantel et al. 2004). The feeding behavior between the different balitorine species is comparable, but both morphological types of the kinethmoid are found. This supports the recent findings of Staab et al. (2012). According to the theory that not only a one-to-one relation is present between an individual morphological trait and the overall function of an integrated system (Alfaro et al. 2007, Wainwright 2007), Staab et al. (2012) then suggested that kinethmoid shape, but also the muscle branching of A1 and the nature of the ligamentous connections are important for jaw protrusion.

The dominant factor determining kinethmoid shape is therefore still unknown. We observed in S. lineolata that, next to the kinethmoid, also the autopalatine is slender. The kinethmoid is connected with ligaments to this structure (Staab et al. 2012) and this connection is highly conserved within Cypriniformes. Therefore, the ligamentous connection with the autopalatine could play an important role to explain kinethmoid morphology.

4.2 Feeding and respiration

Our research has shown that no significant differences were found in the total respiration cycle time between feeding and non-feeding behavior. However, feeding relied on an expiration time that was about twice as long as during non-feeding. It is not known which muscles are exactly responsible for the observed movements but a lot of research has been done on the feeding mechanisms of actinopterygian fish by e.g., Alexander (1966, 1967), Liem (1970) and Lauder (1980, 1982). Ballintijn et al. (1972) performed an electromyographic study on the adductor mandibulae complex of Cyprinus carpio during feeding. We hypothesize that the action of the muscles in the carp is similar to the actions in Balitorinae. However, we found some differences between Balitorinae and C. carpio.

In C. carpio, the adductor mandibulae is subdivided in A1α, A1β, A2, A3 and Aω. The latter muscle bundle is small in the carp and has no important function. We did not observe such a muscle bundle in the serial section of both Beaufortia leveretti and Gastromyzon punctulatus. It was also not possible to clearly distinguish A1α and A1β, but other studies have shown that both are present in several Cypriniformes (Takahasi 1925, Winterbottom 1974, Fink & Fink 1981, Howes 1983a, Gosline 1989).

The lower jaw in carp is elevated by contraction of A3 and this way, the mouth is closed. It also keeps the mouth shut during retraction of the upper jaw while feeding. Contraction of the A2 muscle bundle can increase the speed of lower jaw elevation and is only used during feeding. Rotation of the kinethmoid is caused by contraction of A1β. This rotation leads to the protrusion of the premaxillae and maxillae. The contraction of A1β is usually associated with activity of A1α. This has a threefold function in the carp.

First, by the combined action of A1α and A1β, the upper jaw is protruded and extended downward in such a way that the opening of the mouth is pointed downwards. The lips can thus be applied close to the surface of the bottom when food needs to be sucked in. The mouth of Balitorinae is shifted to a permanent ventral position and thus already lies in close contact with the substrate. Therefore, it seems highly unlikely that such an activity is necessary. No significant differences in upper jaw excursions were measured in our research between feeding and non-feeding conditions, suggesting that such a function is indeed not used in Balitorinae.

Secondly, the action of the two parts of A1 causes a downward extension of the upper jaw so that the mouth is closed without the necessity to elevate the lower jaw. If the lower jaw is elevated when food is in the mouth, the buccal cavity would decrease and food might be swept out of it. By the combined action of A1α and A1β, this is prevented. As we have stated above, the expiration time during feeding is much longer than the inspiration time in Balitorinae. This also means that the mouth is only opened for a short time and the volume of the buccal cavity is only maximum during less than 60 ms. Food particles are sucked in very rapidly into the buccal cavity and no food is observed leaving the mouth. We also observed that the upper jaw indeed started to close the mouth while the lower jaw was still fully depressed during feeding, whereas during normal breathing, both the lower jaw and upper jaw initiate the closure of the mouth simultaneously. Due to this, we speculate that the closing of the mouth, initiated by the upper jaw solely, is caused by the contraction of A1α and A1β.

The third and final function of the combined activity of A1α and A1β in C. carpio is to keep the mouth closed by downward extension of the upper lip during a whole cycle of depression and elevation of the lower jaw. This movement allows the fish to breathe while it is chewing or swallowing food particles without running the risk of losing it. Such observations were, however, not made by us.

Ballintijn et al. (1972) also observed a rare activity of A1α alone when the mouth is completely closed. When the muscle contracts, the upper jaw is retracted further and also the lower jaw is elevated more than usually. Balitorinae, however, use this type of movement as general feeding mechanism. We mentioned that the lower jaw is elevated further, even when the mouth is closed. According to us, this action increases the volume of the cavity below the body. Associated with the skin in front of the upper lip being pulled posterodorsally, this results in a strong suction effect underneath the body. When the mouth is then opened, a low pressure is created in the buccal cavity and water is carried into the mouth due to the pressure differential with the surrounding water, as described by Lauder (1980).

The insertion points of the retractor tentaculi (Fig 15) are unusual in comparison with related taxa. Its relative position in the head is comparable to the position of the musculus rostralis of Lefua costata (Nemacheilinae) in a study of Kim & Kim (2007). According to this study, the musculus rostralis is responsible for barbel movement as connective tissue runs towards the barbel. Therefore, we state that it is homologous to the retractor tentaculi (Fig 15 and 28).

Figure 28: A: Lateral view of the the head muscles, anterior to the left. A: Lefua costata (modified from Kim & Kim (2007)7). B: Beaufortia leveretti.AM: Adductor mandibulae, RT: Retractor tentaculi. Scale bar = 1mm. As the lacrimal extended forward during evolutionary history, the retractor tentaculi could have lost its connection to the barbels and become attached to this lacrimal. The barbels of the studied Balitorinae are strongly reduced or absent. As the retractor tentaculi does not insert on the reduced barbels in the studied Balitorinae, they cannot be moved. We expect that the novel insertion points of the retractor tentaculi allow controlled inspiration. The insertion on the immobile lacrimal acts as an anchor point. When the retractor tentaculi contracts, the skin in front of the upper lip is pulled anteroventrally, and therefore, the skin attaches to the substrate. In concordance with the closure of the mouth, the muscle relaxes, causing the skin to move posterodorsally and to lose its contact with the substrate. This allows water to flow underneath the body, which is then used for respiration. This way, the retractor tentaculi plays a role in the controlled inspiration of Balitorinae. It is therefore comparable in function with the maxillary barbels in Loricariidae (Siluriformes), which are responsible for controlling the inspiration and preventing failure of the sucker system (Geerinckx et al. 2007).

7 Indications removed. ‘Musculus rostralis’ replaced by ‘Retractor tentaculi’ and the different adductor mandibulae subdivisions by ‘AM’.

The hypothesis that no differences are present in the respiration rate when the specimens are in flowing or in still water is supported. The duration of the respiration cycle in both still and flowing water is similar, indicating that the fishes are adapted to torrential life and aren’t significantly influenced by the condition of water flow. However, high overall variation in the duration of the respiration cycle was found. This high variation is probably caused by another factor, such as the oxygen level, stress or the activity of the specimens at the time they were recorded.

We expected changes in respiration rate between feeding and non-feeding behavior. Although no differences in the total respiration rate were found, there was a significant difference in both the inspiration and expiration rate. The inspiration rate was half as long during feeding in comparison to during non-feeding, whereas the expiration rate is significantly longer.

This simultaneously supports our hypothesis that timing differences are present between feeding and non-feeding conditions. When the mouth is closed during feeding, the upper jaw is elevated further, whereas this is not the case during normal breathing. The timing of elevation of the lower jaw thus differs between feeding and non-feeding behavior. We also found a second timing difference. During feeding, the upper jaw starts closing the mouth before the lower jaw elevates. This is in contrast to non-feeding behavior, during which both the upper and lower jaw start closing the mouth at the same time.

We found that the pectoral fins are not used more often when the specimens are in flowing or still water. The use of the pectoral fins in still water could be an indication that attachment is not perfect and that water can leak underneath the body through the gaps between the unculi or along the head region. The fact that pectoral fins are not used more or less frequently in flowing or still water may, however, indicate that the body profile of the species still allows effective attachment to the substrate, preventing extra leakage underneath the body in flowing water. Another possibility is that by facing the flowing water, the body is pressed more effectively to the substrate (see ‘4.3 attachment’) and that extra leakage is prevented this way. In still water, the body would not be pressed so effectively to the substrate causing water to leak more easily underneath the body.

The kinematic analysis showed that the mouth is responsible for most of the water removal. By observing water flow, however, we found that volumes of water can also be removed from underneath the body by detachment of the posterior fin rays of the pectoral fins from the body. Hora (1930) mentioned that the posterior fin rays are constantly in motion in fish with non-overlapping fins using attachment. The fin rays help to pump out leakage water from the ventral surface of the fish when it is perched to the substrate. This would keep the body fixed on the substrate. The fact that the fins are not constantly moving in the investigated Balitorinae might be an indication that their body profile indeed allows effective attachment, preventing extra leakage, as water does not needs to be pumped out continually. It is also possible that Balitorinae can allow small volumes of water underneath their body without running the risk of being detached. If the water volume underneath the body would, however, increase too much, this could lead to detachment from the substrate. Therefore, the posterior fin rays are moved to remove this water, allowing the fish to attach sufficiently on the substrate. The observation that the fins are moving rapidly when the ventral surface of the fish is not fully attached to the substrate suggests that they indeed play an important role in water removal. The pectoral fin thus have a supportive role in attachment behavior, which supports our final hypothesis.

4.3 Attachment

Station-holding (maintaining a certain position in a current) in teleost fish can depend on suction behavior, friction-enhancing structures or both (Gerstner 2007). Our research has shown that the studied balitorine species are evolved in such a way that they are fully adapted to fast-flowing waters.

We already mentioned that a discrimination can be made between two morphological types in Balitorinae. Enlarged, overlapping fins are not found in other cypriniform fishes, indicating that normal-sized fins are the ancestral state. Within Balitorinae, two clades can be distinguished (Tang et al. 2006, Liu et al. 2012). These clades correspond to the former families Gastromyzontidae and Homalopteridae (Silas 1953). The presence of enlarged fins is present in both clades and this character state thus evolved at least twice (Fig 29). From this, it can be deduced that the presence of such fins is beneficial in torrential rivers.

Figure 29: Phylogeny of Balitorinae according to Tang et al. 2006. A: Lepturichthys fimbriata8. B: Beaufortia kweichowensis9. Species in green rectangles exhibit overlapping fins. Balitorinae evolved in such a way that the whole body is used as a sucker, an observation also made by Hora (1930). Their enlarged paired fins allow strong fin-appression. During fin- appression, the anterior part of the pectoral fin is held horizontally and is closely attached to the substrate. The posterior part of the fin curves in dorsal direction, this way, its dorsal surface faces the current. Next to fin-appression, a second type of fin behavior, called fin- standing, was described by Lundberg & Marsh (1976). This type of behavior was not

8 Source: http://www.aquariophil.org/html/lept_fimbriata.html

9 Source: http://www.seriouslyfish.com/species/beaufortia-kweichowensis/

49 observed in the investigated Balitorinae, but is observed in the cypriniform Catostomidae, Psilorhynchidae, Gyrinocheilidae and Balitoridae without overlapping fins. During fin- standing (Fig 30), the pectoral fins are held extended in ventral direction, supporting the anterior part of the body. The most anterior fin rays are longer than the posterior ones and thus have more contact with the substrate. Lundberg & Marsh (1976) observed fin-appression in Moxostoma anisurum and Hypentelium nigricans (Catostomidae, Cypriniformes) at a current of 15 cm/s. They stated that fin-standing would be mainly used in standing and slowly flowing water, while in fin-appression behavior would take place in (fast-)flowing waters.

Figure 30: Fin-standing behavior in Psilorhynchus balitora10. We mentioned that Balitoridae are mainly found in riffles, where high velocity is present (Martin-smith 1998a). Therefore, we suspect that the pectoral fins of the investigated Balitorinae have evolved in function of optimal fin-appression, being able to perch effectively at current speeds of 40 cm/s. The fins are strongly developed and characterized by large pectoral radials, carrying 20 fin rays in total. The anterior fin rays are shorter than the posterior fin rays, which is opposite to the condition optimal for fin-standing. Having elongated fin rays more posteriorly could be beneficial for suction, as this way, the fin can cover a large surface of the substrate, which optimizes strong attachment. The anterior fin rays of the paired fins are covered with unculi on the ventral side, which act like friction- enhancing structures and thus also increase attachment.

10 Source: http://wpedia.goo.ne.jp/enwiki/Psilorhynchus_balitora

The body profile of the investigated Balitorinae itself also plays a crucial role in attachment. The ventral surface of the balitorine species is flat, allowing close contact to the substrate. The elongation and the increased number of fin rays of the pectoral fin allow the fin to overlap the pelvic fin, preventing water to flow underneath the body. The lacrimal bone in the studied species is very well developed, a condition not found in other Cypriniformes (own observation, Ramaswami 1952). The bone is extended forward in Balitorinae, lying in front of the maxilla and premaxilla. The anterior portion of the bone lies ventral. Therefore, it lies in close contact with the substrate and forms the anterior border of the sucker. The close contact of the whole body to the substrate prevents the specimens from being dragged away. We suspect that the body of the fish is pressed against the substrate when facing the water current (Fig 31).

The use of fins and such an evolution of the body profile is quite unique among other hill stream fishes that use attachment behavior, as most other suction behavior depends on attachment of lips and a suction disc or on a combination of the lips and the fins (Hora 1930).

Figure 31: Lateral view of Beaufortia leveretti. Blue arrow: direction of water current. Green arrows: Head is pushed down. The genus Garra (Cyprinidae), for example, is a typical hill stream fish that is in possession of a mental disc as an adhesive apparatus (Saxena & Chandy 1966). This disc is present just posterior to the lower lip and has a circular button-like form (Fig 32). It consists of a central circular callus pad and a free convoluted posterolateral border. This callus pad is covered with tubercles, which are also present in a high density on the anterior lip (Saxena 1959). The stratum corneum of these regions extends and gives rise to spines, which can overgrow the tubercles. Spines are also present on the paired fins of these species.

The body of Garra is not dorsoventrally flattened and does not exhibit the same body profile as the studied Balitorinae. For effective attachment, they thus developed a specialized adhesive apparatus, but they also depend on friction-enhancing behavior.

Figure 32: Ventral view of Garra arupi sp11. Another genus capable of strong attachment is Glyptohorax (Sisoridae, Siluriformes). Saxena & Chandy (1966) performed a study on these species. These fishes are in the possession of a thoracic adhesive apparatus, which is formed by folds of skin arranged in a definite pattern, each fold alternating with a small groove (Fig 33). On these folds and on the paired fins as well, keratinized spines are present (Sinha et al. 1990). According to Hora (1922) and Bhatia (1950), this apparatus operated only on a frictional principle, with the folds preventing the fish from slipping. The study of Saxena & Chandy (1966), however, suggested that due to the pressure on the thoracic adhesive apparatus during adhesion, both water and air could be expelled, creating a partial underpressure in the grooves of the successive folds.

Figure 33: Ventral view of Glyptohorax kurdistanicus12.

11 Source: http://threatenedtaxa.org/ZooPrintJournal/2009/April/Vishwanath_Figs.htm 12 Source: http://www.arkive.org/glyptothorax/glyptothorax-davissinghi/

Glyptothorax has a slender body which is not dorsoventrally flattened. The fish depends on friction-enhancing structures and a specialized adhesive apparatus. The use of friction- enhancing structures and/or an adhesive apparatus is also present in other species that show attachment behavior. Loricariidae (Siluriformes) have a specialized suckermouth and an enlarged pectoral spine ray, which is used as friction-enhancing structure (Geerinckx 2007, 2011). Another example is the development of a ventral sucker in Gobiidae (Perciformes) by the fusion of the pelvic fins (Nelson 2006, Maie et al. 2013).

Important is that all the species mentioned above lack the dorsoventrally flattened body profile of the investigated Balitorinae. The combination of the enlarged, overlapping, paired fins covered with unculi, the dorsoventrally flattened head and body and the flat ventral surface allows Balitorinae to attach effectively to the substrate. Therefore, we suspect that due to the presence of such a body form and profile, effective attachment is obtained without the need of a specialized attachment structure. Instead, the whole body acts as an adhesive apparatus, preventing water to run underneath the body and thus preventing detachment.

For respiration and breathing, water needs to be brought underneath the body. We mentioned that when the skin anterior of the upper lip is pulled posterodorsally, a small volume of water moves underneath the body. Associated with the skin movement, the lower jaw is elevated. This creates an underpressure underneath the body, which could result in an increased suction effect on the water. The stronger elevation of the lower jaw during feeding supports this theory as both food and water can be brought underneath the body very effectively. Most water is removed through the mouth. We have shown that the posterior fin rays, which normally lie in contact with the body, detach to remove the remaining water. The action of these fin rays thus also supports attachment by preventing large volumes of water to run underneath the body.

4.4 Movement

We mentioned that the whole body of the studied Balitorinae is used as a suction unit. During movement, however, it is observed that the body acts as two suckers. The first sucker body bears the pectoral fins, while the second part bears the pelvic fins. In Balitorinae, the two parts are equally important for movement. The anterior sucker moves to the side, due to which one side of the body is brought forward, whereas the posterior part remains attached to the substrate (Fig 24). When the anterior sucker attaches again to the substrate, it is used as an anchor point and the second part is pulled forward. In this way, the movement is comparable to the propulsion of Astroblepidae (Johnson 1912, Shelden 1937, Schaefer & Buitrago-Suarez 2002, De Crop et al. 2013). These species use a suckermouth but also have an enlarged pelvic fin ray. The pelvic fins are pulled forward and then act as an anchor point to draw the body forward. The most important muscles in this process are the retractor and protractor ischii. Whereas contraction of the latter pulls the pelvic fins forward, the drawing forward of the body is caused by contraction of the retractor ischii. These muscles also play an important role in other species using attachment such as the gobies (Maie et al. 2013). We suggest that these muscles could also play a role in the movement of Balitorinae. We observed the contraction of a paired muscle on the ventral side, probably the retractor ischii, during movement. When the anterior part moves to the right (Fig 24B), the right muscle contracts whereas the left muscle is relaxed. This body part then attaches to the substrate and acts as an anchor point. Next, the posterior part of the body moves in the opposite direction with respect to the anterior part (to the left, Fig 24C). Along with this, it is the left muscle that contracts while the right muscle relaxes. Due to these observations, it is thought that movement is partially caused by the consecutive contraction and relaxation of each paired muscle. As no electromyography was applied, this is only a speculation and probably also other muscles, such as the epiaxial and hypaxial muscles, play a role. More and specific research, however, could clarify the question of which specific muscles are involved in movement.

5. Conclusion

The main goal of this research was to describe the internal structures responsible for attachment, feeding and respiration and to understand how they work. We were able to identify the different osteological and myological features. We discovered that the very pronounced enlargement of the lacrimal and the unusual insertion points of the retractor tentaculi are unique for these species and are possible autapomorphies to torrential life. Most structures used for feeding and breathing are similar between the different investigated species. Due to this, the difference in kinethmoid shape between Sewellia lineolata on the one hand and Pseudogastromyzon myersi and Beaufortia leveretti on the other hand is striking and we suggest that the ligamentous connection of this bone to the autopalatine may be an important factor determining kinethmoid morphology. Only speculations could be made of how the different muscles are involved in the actions of feeding, respiration and attachment, based on observations in other species. Due to their small size, no electromyographic study on Balitorinae was performed, but such a study would be helpful in the future to understand how these fishes are specifically adapted to torrential life and to confirm the function of the retractor tentaculi.

We found evidence that all hypotheses were supported. The pectoral fins are supportive in removing water from underneath the body, which allows Balitorinae to attach effectively to the substrate and to create an underpressure between the body and the substrate. Next to this, no significant difference in respiration rate is present between breathing in still or flowing water. Total respiration time is also similar between feeding and non-feeding conditions. The expiration time during feeding is, however, significantly longer in comparison to non-feeding, whereas inspiration time was significantly shorter. Finally, also the hypothesis about timing differences between feeding and non-feeding behavior was supported. It was found that during feeding, the upper jaw initiates mouth closure alone, whereas during non-feeding, the upper and lower jaw close the mouth simultaneously. The lower jaw also elevates further during feeding.

Torrential life is a difficult environment to survive and fishes have adapted in several ways to these circumstances. According to this research, the investigated Balitorinae exhibit three specific adaptations to life in fast-flowing waters: (1) the enlarged lacrimal, (2) the unusal insertion points of the retractor tentaculi and (3) the unique body profile with overlapping, wing-like paired fins covered with unculi, the dorsoventrally flattened body and the flat

55 ventral surface. This specific morphology is not found in other Cypriniformes and is thus unique for the investigated species and morphologically similar Balitorinae that weren’t studied. Species living in fast-flowing rivers that do not exhibit such a body profile developed a specific adhesive apparatus. Multiple possible adhesive structures were mentioned:

- Suckermouth of Loricariidae (Siluriformes) - Suckermouth and pelvic fin apparatus of Astroblepidae (Siluriformes) - Mental adhesive apparatus of the genus Garra (Cyprinidae, Cypriniformes) - Thoracic adhesive apparatus of Glyptothorax (Sisoridae, Siluriformes) - The pelvic sucker of Gobiidae (Perciformes)

It can thus be concluded that, depending on the ancestor-related morphological constraints, natural selection can allow different possibilities to adapt to similar environments.

6. Acknowledgements

I would like to start with thanking my tutor, Tom Geerinckx. By his enthusiasm, advice, insights and the sharing of his experiences, he was the most important help in the writing and completing of this master’s thesis. Next to Tom, I want to thank Barbara De Kegel and Joachim Christiaens, for providing the serial sections of both Beaufortia leveretti and Gastromyzon punctulatus and giving their advice for clearing and staining the other specimens. I also express my thanks to the Florida Museum of Natural History for providing the Lefua costata specimens. My gratitude goes to the research group ‘Evolutionary Morphology of Vertebrates’ itself for the use of their material. I am also indebted to professor Dominique Adriaens and the staff members of the research group: Mathias Bouilliart, Tim tkint, Céline Neutens and Thuong Nguyen Phuc. They were always ready to answer my questions and provided a nice working atmosphere. Also my family and my girlfriend, Laura Van den Berghe, are thanked for their constant support and motivation. Finally, I would like to thank my fellow students, with special reference to Catherine Mermans, for her advice in making understandable and clear drawings and the many laughs we shared.

7. Summary – Samenvatting

7.1 Summary

Functional morphology studies the morphology of structures such as muscles and bones and tries to link this to their function. To understand how the morpholrogy of structures is related to their function, it is useful to study specialized species, characterized by pronounced or extreme structures. Fast flowing rivers are a harsh environment for fishes, because they can easily be swept away by the strong current. To survive in such circumstances, fishes developed specialized morphological modifications. These modifications are mainly manifested in the form of adhesive structures to prevent detachment. One of the families that developed adhesive structures are Balitoridae (Cypriniformes, Teleostei). This research focuses on the subfamily Balitorinae, characterized by an anteriorly flattened body and restricted gill openings, placed above the pectoral fins. They have a ventrally oriented mouth. The pectoral and pelvic fins are inserted low and horizontally and have been expanded so they can be used as sucker-like adhesive organs.

In this research, the balitorine species Sewellia lineolata, Beaufortia leveretti, Gastromyzon punctulatus and Pseudogastromyzon myersi were investigated to describe and identify the different muscles and osteological structures responsible for attachment, feeding and respiration mechanisms. To understand how these structures are used, a kinematic analysis was performed. The kinematic analysis allowed us to find out if there are differences in the mechanisms used in still or flowing water on the one hand and during feeding and non- feeding behavior on the other hand. Four hypotheses were proposed for the kinematic analysis. (1) The same respiration rate is retained in still and flowing water. (2) There is a difference in the respiration rate between feeding and non-feeding conditions. (3) There are timing differences in the activity of the different structures used during feeding and non- feeding. (4) Balitorinae use their pectoral fins to remove water from underneath the body to enhance attachment. Finally, the gathered data was compared with those of related taxa and other species using attachment, to find out whether multiple solutions are possible for attachment or whether examples of convergent evolution are present. This allows us to find out how Balitorinae adapted to their environment.

For the kinematic analysis, recordings were made of S. lineolata, B. leveretti and P. myersi. At least three respiration cycles were recorded in still and flowing water at two independent moments per specimen in order to have at least six respiration cycles in each condition. This was done in ventral, dorsal and lateral view. The fishes were also recorded during feeding. Finally, diluted milk was used to visualize water flow. From these recordings, different points of interest were digitized (upper lip, lower lip, tip of the snout, hyoid, operculum, the skin between the head and the pectoral fin and the pectoral fin itself). These data were then used to make graphs for determining the respiration cycle time of these structures. For the identification of the osteological structures, specimens of S. lineolata, B. leveretti and P. myersi were cleared and stained according to the protocol of Hanken & Wassersug (1981). Also a CT scan was made of a B. leveretti specimen. Finally, 5 µm sections were made of B. leveretti and G. punctulatus, for the identification of the muscles.

The most important finding of the osteological research is the strongly enlarged lacrimal bone, which extends anterior of the premaxilla and maxilla. Next to this, the pronounced difference in kinethmoid morphology between B. leveretti and P. myersi on the one hand and S. lineolata on the other hand is striking. While the bone is long and slender in the latter, it is short and broad in the other species. We suspect that the ligamentous connection to the autopalatine may play an important role in determining kinethmoid morphology. Both a first and second preethmoid are present in the investigated species, a feature that is not observed in related taxa (Botiinae (Cobitidae), Nemacheilinae (Balitoridae)). Also the pectoral fins are strongly developed in Balitorinae. The pectoral radials are enlarged, allowing the attachment of 20 fin rays. The anterior fin rays themselves are covered with unculi on the ventral side, which act as friction-enhancing structures.

The head muscles of the studied Balitorinae are similar to those of other related (sub)families. The insertion points of the retractor tentaculi are, however, unique. The muscle inserts on the ventromedial surface of the lacrimal and originates on fat tissue underlying the ventral side of the body, lateral to the dentary. We expect that the retractor tentaculi controls the inflow of water underneath the body and thus allows controlled inspiration.

The kinematic analysis showed that no significant difference in respiration rate was present when the studied Balitorinae were in flowing or still water, indicating that they are effectively adapted to fast-flowing rivers. No significant difference in total respiration rate was found between feeding and non-feeding behavior either. During feeding, however, the inspiration

59 time was significantly shorter in comparison to non-feeding, whereas the expiration time was significantly longer. The increased expiration time during feeding is associated with a further elevation of the lower jaw. We expect that this creates a larger underpressure underneath the body. This larger underpressure allows a stronger suction effect of water under the body. It was also found that during feeding, the upper jaw starts closure of the mouth while the lower jaw remains depressed, preventing food particles to escape from the mouth. During breathing, however, closing of the mouth is initiated by the lower and upper jaw simultaneously.

We found that the studied Balitorinae are effectively adapted in function of strong attachment. The flat ventral surface of the body, the enlarged fins and the flattened head with the enlarged lacrimal allow close contact to the substrate and thus the whole body acts as an adhesive apparatus. We suspect that the fish is pressed to the substrate when facing the water current due to this close contact. Inflow of water underneath the body from the lateral sides is prevented by the enlarged pectoral and pelvic fins with the pectoral fins overlapping the latter. Our kinematic analysis showed that water entering the cavity between the body and the substrate along the head region and through the gaps between the spines of the fins is removed through the mouth and by the posterior fin rays of the pectoral fins. These fin rays are usually attached to the body. When there’s too much water underneath the body, they detach, creating a gap between the body and the fin. The water underneath the body is then removed through this gap, indicating a supportive role of the pectoral fins in attachment. This supports our final hypothesis.

The use of the whole body as a suction apparatus is quite unique for attachment behavior, as other taxa developed more specific adhesive structures, e.g. the suckermouth of Loricariidae and Astroblepidae (Siluriformes) or the mental sucker of Garra (Cyprinidae, Cypriniformes) and and thoracic sucker of Glyptothorax (Sisoridae, Siluriformes). Due to the specialized body profile of Balitorinae, no such structure is necessary for effective attachment. From this, it can be concluded that natural selection can allow different possibilities for species to adapt to similar environments, depending on the ancestor-related morphological constraints.

7.2 Samenvatting

Functionele morfologie bestudeert de morfologie van structuren zoals spieren en beenderen en probeert dit te linken aan de functie van de structuren. Om te begrijpen hoe de morfologie van een structuur gerelateerd is aan hun functie is het nuttig om gespecialiseerde soorten te bestuderen, die gekarakteriseerd worden door uitgesproken of extreme structuren. Snelstromende rivieren vormen een moeilijke omgeving voor vissen omdat ze door de sterke stroming makkelijk meegesleurd kunnen worden. Om te overleven in zulke omstandigheden hebben ze gespecialiseerde morfologische modificaties ontwikkeld. Deze modificaties worden meestal gemanifesteerd onder de vorm van aanhechtingsstructuren die loshechting voorkomen. Balitoridae (Cypriniformes, Teleostei) is een van de families die aanhechtingsstructuren ontwikkelden. Dit onderzoek focust op de subfamilie Balitorinae, die gekarakteriseerd worden door een afgeplat lichaam en beperkte kieuwopeningen boven de pectorale vinnen. Hun mond is ventraal georiënteerd. De pectorale en pelvische vinnen zijn laag en horizontaal geplaatst en zijn zodanig uitvergroot dat ze gebruikt kunnen worden als zuigachtige aanhechtingsorganen.

In dit onderzoek werden de soorten Sewellia lineolata, Beaufortia leveretti, Gastromyzon punctulatus en Pseudogastromyzon myersi onderzocht om de verschillende spieren en beenderen die gebruikt worden voor voeding, ademhaling en aanhechting te identificeren en te beschrijven. Om te begrijpen hoe deze structuren gebruikt worden, werd een kinematische analyse uitgevoerd. Deze analyse stond ons toe om te ontdekken of er verschillen zijn in de mechanismen die gebruikt worden in stilstaand of stromend water enerzijds en de mechanismen gebruikt gedurende voeding en niet-voeding anderzijds. Vier hypotheses werden opgesteld voor deze analyse. (1) De ademhalingstijd is gelijk in stilstaand en stromend water. (2) Er is een verschil in ademhalingstijd tussen voedingsmechanismen en niet-voedingsmechanismen. (3) Er zijn verschillen in de timing van de activiteit van de verschillende structuren gedurende voeding en niet-voeding. (4) Balitorinae gebruiken hun pectorale vinnen om water onder het lichaam te verwijderen en om op deze manier aanhechting te verbeteren. Ten slotte werd de verzamelde data vergeleken met gerelateerde taxa en andere soorten die aanhechting gebruiken. Dit stond ons toe om te ontdekken hoe Balitorinae aangepast zijn aan hun omgeving.

Voor de kinematische analyse werden opnames gemaakt van S. lineolata, B. leveretti en P. myersi. Er werden minstens drie ademhalingscycli in stilstaand en stromend water opgenomen per specimen en dit op twee onafhankelijke tijdstippen. Op deze manier werden op zijn minst zes ademhalingscycli gefilmd. Dit gebeurde in ventraal, dorsaal en lateraal zicht. Er werden ook opnames gemaakt terwijl de vissen aten. Tot slot werd verdunde melk gebruikt om de waterstroming te visualiseren. Verschillende punten (bovenlip, onderlip, tip van de snuit, hyoid, operculum, de huid tussen de kop en de pectorale vin en de pectorale vin zelf) werden gedigitaliseerd in deze opnames. Van de bekomen data werden vervolgens grafieken gemaakt opdat de cyclustijd van deze structuren kon bepaald worden. Voor de identificatie van de osteologische structuren werden specimens van S. lineolata, B. leveretti en P. myersi opgehelderd en gekleurd volgens het protocol van Hanken & Wassersug (1981). Er werd tevens een CT scan genomen van een B. leveretti specimen. Ten slotte werden 5µm dikke secties gemaakt van B. leveretti en G. punctulatus om de spieren te identificeren.

De belangrijkste ontdekking van het osteologisch onderzoek was het vinden van het sterk vergrootte lacrimale, die tot voor de premaxilla en maxilla reikt. Daarnaast werd een opvallend verschil gevonden in de vorm van het kinethmoid tussen B. leveretti en P. myersi enerzijds en S. lineolata anderzijds. Het kinethmoid is lang en smal in S. lineolata, terwijl het breed en kort is in de andere soorten. We vermoeden dat de ligamenteuze connectie met het autopalatinum een belangrijke rol speelt in het bepalen van de kinethmoid morfologie. Zowel het eerste als tweede preethmoid zijn aanwezig in de onderzochte soorten, terwijl deze niet gevonden worden in de gerelateerde taxa (Botiinae (Cobitidae), Nemacheilinae (Balitoridae)). Ook de pectorale vinnen zijn sterk ontwikkeld in Balitorinae. De pectorale radialen zijn vergroot en deze vergroting staat de aanhechting van 20 vinstralen toe. De ventrale zijde van de voorste vinstralen is bedekt met unculi, die frictie verbeteren.

De kopspieren van de onderzochte Balitorinae zijn vergelijkbaar met die van gerelateerde (sub)families. De insertieplaatsen van de retractor tentaculi zijn echter uniek. De spier hecht rostraal aan op het ventromediale oppervlak van het lacrimale en caudaal op vetweefsel aan de ventrale zijde van het lichaam, lateraal van het dentale. We speculeerden dat de spier de instroom van water onder het lichaam controleert en aldus gecontroleerde inspiratie toelaat.

De kinematische analyse toonde aan dat er geen significant verschil is in de ademhalingsratio wanneer de bestudeerde Balitorinae zich in stromend of stilstaand water bevonden, wat erop wijst dat deze dieren effectief aangepast zijn aan snelstromende rivieren. Er is ook geen significant verschil in de totale ademhalingstijd tussen voeding en niet-voeding. Gedurende het eten duurde de inademing significant korter dan gedurende normaal ademhalen, terwijl de uitademing significant langer was. Deze verlengde uitademing is geassocieerd met een verdere elevatie van de onderkaak, waarvan we vermoeden dat het een grotere onderdruk creëert onder het lichaam. Door deze vergrootte onderdruk kan een sterkere zuigkracht uitgeoefend worden op het water. Er werd ook geobserveerd dat gedurende het eten de bovenkaak begint met het sluiten van de mond, terwijl de onderkaak omlaag blijft, waardoor voedselpartikels niet uit de mond kunnen ontsnappen. Gedurende ademhaling gebeurt het sluiten van de mond echter simultaan door de onder- en bovenkaak.

Het onderzoek wees uit dat de bestudeerde Balitorinae aangepast zijn in functie van sterke aanhechting. Het platte, ventrale oppervlak van het lichaam, de vergrootte vinnen en de platte kop met het vergrootte lacrimale staan nauw contact met het substraat toe en het hele lichaam wordt dus gebruikt als aanhechtingsorgaan. We vermoeden dat de vis tegen het substraat wordt geduwd door dit nauwe contact en de blootstelling aan de snelle stroom. Instroom van water onder het lichaam langs de laterale zijden wordt vermeden door de overlappende, gepaarde vinnen. Onze kinematische analyse toonde aan dat water dat onder het lichaam terechtkomt langs de kopregio en doorheen de holtes tussen de unculi op de vinnen, verwijderd wordt door de mond en de achterste vinstralen van de pectorale vinnen. Deze vinstralen, die gewoonlijk aan het lichaam hechten, komen los waardoor er een holte gevormd wordt tussen het lichaam en de vin. Het water onder het lichaam wordt vervolgens verwijderd langs deze holte, wat wijst op een ondersteunende functie van de pectorale vinnen voor effectieve aanhechting. Dit bevestigde de laatste hypothese.

Het gebruik van het hele lichaam als zuigapparaat is uniek als aanhechtingsgedrag, aangezien andere taxa meer gespecialiseerde aanhechtingsstructuren ontwikkelden, zoals de zuigmond van Loricariidae en Astroblepidae, de kinzuignap van Garra (Cyprinidae) en de borstzuignap van Glyptohorax (Sisoridae, Siluriformes). Door het gespecialiseerde lichaam van Balitorinae is het niet nodig dat zo’n structuur gevormd wordt. Er kan hieruit dus besloten worden dat natuurlijke selectie verschillende mogelijkheden toelaat voor soorten om zich aan te passen aan gelijkaardige omgevingen, afhankelijk van de morfologische beperkingen van hun voorouders.

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