Vertebral column regionalisation in Chinook salmon, Oncorhynchus tshawytscha

Abstract

Teleost vertebral centra are often similar in size and shape, but vertebral‐associated elements, i.e. neural arches, haemal arches and ribs, show regional differences. Here we examine how the presence, absence and specific anatomical and histological characters of vertebral centra‐associated elements can be used to define vertebral column regions in juvenile Chinook salmon (Oncorhynchus tshawytscha). To investigate if the presence of regions within the vertebral column is independent of temperature, animals raised at 8 and 12 °C were studied at 1400 and 1530 degreedays, in the freshwater phase of the life cycle. Anatomy and composition of the skeletal tissues of the vertebral column were analysed using Alizarin red S whole‐mount staining and histological sections. Six regions, termed I–VI, are recognised in the vertebral column of specimens of both temperature groups. Postcranial vertebrae (region I) carry neural arches and parapophyses but lack ribs. Abdominal vertebrae (region II) carry neural arches and ribs that articulate with parapophyses. Elastic‐ and fibrohyaline cartilage and Sharpey's fibres connect the bone of the parapophyses to the bone of the ribs. In the transitional region (III) vertebrae carry neural arches and parapophyses change stepwise into haemal arches. Ribs decrease in size, anterior to posterior. Vestigial ribs remain attached to the haemal arches with Sharpey's fibres. Caudal vertebrae (region IV) carry neural and haemal arches and spines. Basidorsals and basiventrals are small and surrounded by cancellous bone. Preural vertebrae (region V) carry neural and haemal arches with modified neural and haemal spines to support the caudal fin. Ural vertebrae (region VI) carry hypurals and epurals that represent modified haemal and neural arches and spines, respectively. The postcranial and transitional vertebrae and their respective characters are usually recognised, but should be considered as regions within the vertebral column of teleosts because of their distinctive morphological characters. While the number of vertebrae within each region can vary, each of the six regions is recognised in specimens of both temperature groups. This refined identification of regionalisation in the vertebral column of Chinook salmon can help to address evolutionary developmental and functional questions, and to support applied research into this farmed species.

Introduction

The teleost vertebral column is a complex structure comprising building blocks (vertebral centra) of relatively similar size and shape in most species. There is agreement about the presence of three major anterior–posterior regions within the vertebral column. Region one comprises the vertebral bodies from the occipital region of the cranium to the last rib‐bearing vertebral body. Region two consists of haemal arch‐bearing vertebral bodies. Region three is characterised by the vertebral bodies of the caudal fin complex. Different anatomical terms have been used for these regions (Nybelin, 1963; Arratia et al. 2001; Bird & Mabee, 2003; Deschamps et al. 2008; Sallan, 2012). Terms used in the literature for region one are precaudal, abdominal, truncal or prehaemal. In this region some authors distinguish also postcranial (Deschamps et al. 2008) or cervical vertebral bodies (Ford, 1937; Morin‐Kensicki et al. 2002; Holley, 2007; Fjelldal et al. 2013). Terms used for region two are caudal or haemal. Terms used for vertebrae of region three are postcaudal or ural (Nybelin, 1963; Cubbage & Mabee, 1996; Deschamps et al. 2008; Britz & Conway, 2009; Koumoundouros, 2010; Sallan, 2012). Several authors have recognised a region with transitional vertebral bodies between region one and two (Balfour & Parker, 1882; Bird & Mabee, 2003; Holley, 2007; Nowroozi et al. 2012; Jawad et al. 2013), but little or no attention has been paid to this region so far.

Teleosts have different elements associated with the vertebral centra, such as parapophyses, ribs and neural and haemal arches, of very different sizes and shapes (Arratia et al. 2001). The types of associated elements, as well as their development and homology have been extensively described and discussed in Balfour & Parker (1882), Gadow & Abbott (1895), Arratia & Schultze (1992), and Arratia et al. (2001). Some authors have used the associated elements attached to the vertebral centra as characteristics to characterise the regions of the vertebral column. For Oncorhynchus species, for example, Gill & Fisk (1966) subdivided the vertebral column of sockeye, pink and chum salmon (O. nerka, O. gorbuscha, O. keta, respectively) into an abdominal and caudal region. In this system the presence of the first haemal arch defines the first caudal vertebra. In comparison, Seymour (1959) suggests a regionalisation of the vertebral column of Chinook salmon (O. tshawytscha) in which the presence of the first haemal spine defines the beginning of the caudal region. Other characteristics that display anterior to posterior alterations are skeletal tissue components and how the vertebrae‐associated elements are connected to centra. Together these characters provide insight into regionalised vertebral column anatomy.

Regionalisation of the vertebral column is important from a functional, evolutionary and applied perspective. Which regions are recognised largely depends on the research approach used. To compare the structural and functional components of the vertebral column within teleosts (e.g. swimming modes of different fish species), a clear distinction of regions of the vertebral column is necessary. Ramzu & Meunier (1999) used morphometric characters of vertebral centra along the axis of rainbow trout (Oncorhynchus mykiss) to distinguish several functional regions within the vertebral column related to mode of swimming. In another approach, regions are recognised and interpreted based on elongation of fish throughout evolution. For example, the change in ratio of abdominal to caudal vertebrae is interpreted on an evolutionary time scale (Ward & Brainerd, 2007) and subdivides the vertebral column only into major anterior and posterior regions. A study investigating the meaning of the presence of vestiges, rudiments and fusions in the evolution of the caudal fin skeleton of zebrafish (Bensimon‐Brito et al. 2012) focuses only on the posterior‐most part of the axial skeleton. Understanding of aetiologies that cause deformities of the vertebral column in farmed fish is another application for a detailed vertebral column subdivision. Compression of vertebral centra in Atlantic salmon (Salmo salar) occurs in defined and functionally relevant regions, such as the caudal and ural regions (Wargelius et al. 2005; Fjelldal et al. 2009; Grini et al. 2011). The occurrence of deformed vertebrae in the caudal and ural regions affects the swimming behaviour of farmed salmon (Fjelldal et al. 2012). Severely deformed fish constitute a problem for animal welfare and for animal growth (Fjelldal et al. 2012). A refined identification of vertebral column regions integrating different sets of morphometric, anatomical and molecular characteristics could help to better understand the normal development and development of deformities within the vertebral column of farmed salmonids, and could provide better descriptions of how deformities progress through freshwater and marine life stages (Witten et al. 2006; Grini et al. 2011; Fjelldal et al. 2012). Molecular studies have found that subdivision of most parts of the mammalian vertebral column corresponds to boundaries of Hox‐gene expression (Burke et al. 1995; Galis, 1999; Asher et al. 2011). However, Hox‐genes appear to be only partly responsible for the regionalisation of the vertebral column in zebrafish, i.e. the Hox‐genes anterior expression boundaries are concentrated in the anterior‐most part of the vertebral column (Morin‐Kensicki et al. 2002; Holley, 2007).

Here the vertebral column of juvenile Chinook salmon, O. tshawytscha is studied. The species was introduced to New Zealand between 1901 and 1907 for game fishing (McDowall, 1994). Chinook salmon established a wild population on the east coast of New Zealand's South Island by 1915, and became a species of interest for commercial salmon farming in 1970 (Unwin & Glova, 1996). River water temperatures in New Zealand range from 6 °C to 16 °C (Davis & Unwin, 1989; Unwin & Glova, 1996; Kinnison et al. 1998; Unwin et al. 2000) with preferred incubation temperatures of Chinook salmon ranging from a minimum 3 °C to a maximum 12 °C, and optimum temperature range from 8 °C to 10 °C (Brett, 1952; Alderdice & Velsen, 1978). Temperatures near the optimum and high end of the incubation range are more likely used in farming conditions to induce fast growth (Fjelldal et al. 2012). This study asks how the presence or absence, and specific anatomical and histological characters of vertebral centrum‐associated elements can be used to define different regions in the vertebral column of Chinook salmon. To this end, region‐specific changes of vertebral centrum‐associated elements along the vertebral column are characterised. Furthermore, to assess whether the characters of the regions within the vertebral column are temperature dependent, we use specimens raised at two temperatures (8 °C and 12 °C). This information is used to establish: (i) six different vertebral types characteristic for six different regions; and (ii) four vertebral subtypes.

Materials and methods

Fish maintenance and sampling

Approximately 60 000 Chinook salmon eggs obtained from 10 females were randomly divided over 10 batches. The batches were artificially fertilised (20/05/2014) with milt obtained from six males (brood stock provided by New Zealand King Salmon Co. Ltd., Nelson, New Zealand). Fertilised eggs were placed into two incubation stacks. Each incubation stack (seven incubation trays/stack, ± 4000 eggs/incubation tray) was initially supplied with water at a constant 10 °C from fertilisation to 3 days post‐fertilisation (= 30 dd, degreedays). Subsequently the temperature was gradually adjusted over 24 h to constant 8 °C and 12 °C rearing temperatures. Dead individuals were removed once a week during the incubation period before hatching (175–475 dd), every 2 days during the hatching period (475–590 dd), and twice a week after hatching (590–900 dd) to avoid fungal infections and reduce mortalities. At first feed stage 4000 fish from the 8 °C (933 dd) and 12 °C (943 dd) groups were transferred to four 420‐L circular tanks (two tanks per temperature). The fish were fed manually to satiation every hour during the day. Fish were randomly sampled at 1400 and 1530 dd for each temperature, and killed by anaesthetic overdose of Aqui‐S (New Zealand, Ltd.). At sampling 50 specimens were taken from each of the four tanks, fixed in 4% buffered paraformaldehyde and stored in 60% ethanol.

Whole‐mount staining

In total 120 fish were stained for mineralised bone with Alizarin red S (N = 30 at 1400 dd and N = 30 at 1530 dd for 8 °C and 12 °C groups) following an adapted protocol of Taylor & Van Dyke (1985). Briefly, after weighing and measuring fork length (FL), specimens were rehydrated, bleached (1% KOH/3% H₂O₂, 1–1.5 h), rinsed in demineralised water and placed for 24 h in a 70% saturated borax solution (di‐sodium tetraborate). Fish were stained overnight in a 0.5 mg/mL Alizarin red S/1% KOH solution and rinsed in demineralised water. Specimens were cleared in graded glycerol/KOH series, dehydrated in graded glycerol series and brought to 100% glycerol storage solution. Skeletal structures were analysed using a stereomicroscope (Leica M80).

Histology

Four juvenile specimens were used for histology. Two specimens of 1400 dd (8 °C and 12 °C, respectively) and two specimens of 1530 dd (8 °C and 12 °C, respectively) were used. The 1400 dd specimens weighed 1.38 and 3.09 g, and had FLs of 46.36 and 62.26 mm, respectively. The 1530 dd specimens weighed 1.61 and 5.88 g, and measured 57.28 and 75.32 mm, respectively. Tissue blocks of the postcranial/abdominal region, the transitional region and the entire caudal fin were processed for paraffin embedding and sectioning. Before processing the tissues were placed overnight in acetone to remove fat.

All tissue samples were rinsed in demineralised water for 24 h and subsequently decalcified for 48 h in Morse solution [Morse, 1945; 22.5% formic acid (CH₂O₂; Merck KGaA, Darmstadt, Germany)] and 10% sodium citrate (C₆H₅Na₃O₇*2 H₂O; Merck KGaA, Darmstadt, Germany) and rinsed again, processed overnight and embedded in paraffin. The tissue blocks were either embedded to make parasagittal or transverse sections of 4 μm on a Leica RM 2235 microtome. Sections were stained with haematoxylin and eosin (adapted from Gill et al. 1974), Masson's Trichrome and Verhoeff‐Van Gieson methods to highlight collagen and elastin (Culling, 1974a,b). Sections were analysed with brightfield under a Zeiss Axiophot microscope. Images were taken with an Olympus DP72 top mount camera. Illustrator CS5 was used to draw schematics.

Terminology

Structures of the vertebral centrum and the vertebral centrum‐associated elements are defined in Table 1, following Schultze & Arratia (2013) and other authors listed in the table. Cartilages of the vertebral centra and associated elements were defined following the classification of Witten et al. (2010). The types of bone were defined following Hall (2015a, p. 4), and Sharpey's fibres following Hall (2015b, p. 140) and Witten & Hall (2002, 2003, 2015). Finally, following Hall (2003) a vestige is defined as a remnant of an ancestral feature that persists in adults, and a rudiment as an incomplete transitory structure that is only found in embryos.

Table 1.

Definition of terms used

Term	Definition	Reference
Basidorsal	The dorsally positioned cartilaginous part of the neural arch resting with the base on the external elastic membrane of the notochordal sheath	Gadow & Abbott (1895)
Basiventral	The ventrally positioned cartilaginous part of the haemal arch resting with the base on the external elastic membrane of the notochordal sheath	Gadow & Abbott (1895)
Cancellous bone	Consists of different combinations of curved plates with holes of various sizes, and a latticework of rods of a variety of lengths and cross‐sectional diameters. Trabecula is an individual structural element of cancellous bone tissue, whether plate‐like or rod‐like	Parfitt (1988), Whitehouse (1974)
Epicentral	Intermuscular bone ligamentously connected to the cancellous layer of the vertebral centum	Bird & Mabee (2003), Patterson & Johnson (1995)
Epineural	Mineralised intermuscular tendon	Danos & Ward (2012)
Epural	Modified neural arch of ural vertebra	Schultze & Arratia (2013)
Hypural	Modified haemal arch or spine of ural vertebra supporting caudal fin rays	Stiassny (2000)
Parapophysis	Bony structure providing attachment to muscles and articulation for ribs	Owen (1848)
Post‐zygapophysis	Dorsal (neural) or ventral (haemal) extension from the anterior end of a neural or haemal arch. It articulates with the pre‐zygapophysis of the preceding centrum	Bird & Mabee (2003)
Preural vertebrae	Carry slightly modified haemal and neural arches that support caudal fin rays. Haemal arches enclose the caudal artery and vein	Arratia & Schultze (1992), Nybelin (1963), Sanger & McCune (2002)
Pre‐zygapophysis	Dorsal (neural) or ventral (haemal) extensions from the anterior end of a neural or haemal arch. They articulate with the post‐zygapophyses of the preceding centrum	Bird & Mabee (2003)
Rib	Ventral rib, pleurapophysis or haemopophysis are synonyms. Rod‐like bony structure with a head that articulates with the parapophysis	Bird & Mabee (2003), Britz & Bartsch (2003), Gadow & Abbott (1895), Owen (1848)
Stegural	A modified uroneural positioned dorsally of preural 2 and 1, and ural 1 and 2	Arratia & Schultze (1992)
Ural vertebrae	Carry modified neural arches and spines, and modified haemal arches	Schultze & Arratia (2013)
Uroneural	Modified neural spine of ural vertebrae supporting caudal fin rays	Schultze & Arratia (2013)

Results

The same six regions, termed regions I–VI, can be distinguished in the vertebral column in juvenile Chinook salmon (N = 120) of the two temperature groups: the postcranial, abdominal, transitional, caudal, preural and ural regions (Figs 1, 2, 3, 4; Table 2). Temperature does not affect the anatomical subdivision of the regions or the total number of vertebrae (V) ranging from 62 to 68 in both temperature groups. The skeletal structures that show variation are briefly summed up at the end of each section describing the regions. Temperature has possibly a small effect on the number of vertebrae in different regions. A number of morphological characters are common for vertebrae in all regions. Each of these six regions is characterised by vertebrae with a specific array of associated elements (Table 2). Numbers of vertebrae within each region display minor variations. Vertebral centra in all regions have two basidorsals on the dorsal side and two basiventrals on the ventral side (Fig. 1). Basidorsals and basiventrals are composed of cell‐rich hyaline cartilage and connect directly to the bone of the neural and haemal arches, except in regions V and VI. All vertebral centra carry neural arches and neural spines dorsally, except in region VI (Fig. 1). Epineurals are attached to the base of neural arches of regions I and II. At the level of the base of the neural and haemal arch, pre‐ and post‐zygapophyses are present with specific shapes in regions I–II and III–IV. Ribs are present in regions II and III, and are preformed in cartilage, except in region III (subtype IIId). Cartilaginous basidorsals and basiventrals are perichondrally ossifying, except in regions V and VI. Vertebral centra‐associated elements such as parapophyses, ribs, neural arches of type I–IV and haemal arches of type IV vertebrae are completely ossified. Remnants of the cartilaginous precursor are still present in some parapophyses and ribs of type I, II and III vertebrae. The neural spines of type I–IV and haemal spines of type IV vertebrae are intramembranous ossifications. The cartilaginous neural and haemal arches and modified spines of type V vertebrae are perichondrally ossifying. The stegural, epurals and uroneurals dorsally and hypurals ventrally in region VI are perichondrally ossifying.

Figure 1.

Schematic representation of vertebral centra and associated elements per vertebra type (type I–VI). Simplified scheme of vertebral centra types and vertebral centra‐associated elements in the six regions of the vertebral column of juvenile Chinook salmon (± 70 mm). Colours used for different tissue types are indicated in the key. Type I = postcranial; type II = abdominal; type III = transitional; type IV = caudal; type V = preural; and type VI = ural. ant S, anterior spine; BD, basidorsal; BV, basiventral; E, epural; HA, haemal arch; HS, haemal spine; Hy, hypural; NA, neural arch; NS, neural spine; pap, parapophysis; pre‐zyg, pre‐zygapophysis; pst‐zyg, post‐zygapophysis; R, rib; ST, stegural. Arrows on type I and II point to pre‐ and post‐zygapophyses.

Figure 2.

Chinook salmon vertebral column regionalisation, the transitional region and the caudal fin endoskeleton. (A) Simplified scheme of juvenile Chinook salmon vertebral column indicating six types of vertebrae (see Table 2). The transitional region (plus two abdominal vertebrae anteriorly and two caudal vertebrae posteriorly) and the caudal fin endoskeleton (preural and ural vertebral centra and associated elements) are highlighted in the black boxes and presented in more detail in (B) and (C), respectively. Images of areas enclosed in boxes labelled 3D, 3E and 3G are presented in Fig. 3. Orientation of all drawings follows the anterior–posterior (A–P) and dorsal–ventral (D–V) axis indicated in the key. (B) Schematic representation of a typical transitional region based on mean number of vertebrae of 120 Alizarin red‐stained specimens. Dorsally, no distinction is made between the neural arches and neural spines, which are fused to each other. The anterior‐most vertebral centrum in the transitional region has a parapophysis that shows only small differences compared with parapophyses of the last abdominal (type II) vertebrae. The ribs in these transitional vertebrae are attached in a similar way as on abdominal vertebrae (type IIIa). Anteriorly, the parapophyses extend ventrally and form an open haemal arch (type IIIa, Table 2). The ribs are loosely attached to the haemal arches and are in a more postero‐vental position. In type IIIb haemal arches are closed and the bases fuse to the cancellous bone of the vertebral centrum. The bases of the neural arches similarly fuse with the cancellous bone of the vertebral centra. Type IIIc vertebrae have an additional haemal spine that grows longer in each more posterior vertebral centrum. The rib size remains more or less the same. The ribs become vestigial in type IIId (Table 2) vertebrae, and neural and haemal pre‐ and post‐zygapophyses become apparent. BD, basidorsal; BV, basiventral. (C) Schematic representation of the caudal fin endoskeleton, including one caudal vertebra (type IV), preural vertebrae (PU, type V), ural vertebrae (type VI), and their respective associated elements. The cartilage of the basidorsals (BD) of the preural vertebrae is continuous with the cartilage of the neural arches (e.g. NA PU 4); likewise, the cartilage of the basiventrals (BV) is continuous with the haemal arches (e.g. HA PU 4), neural spines (e.g. NS PU 4) and haemal spines (e.g. HS PU 2). The stegural (ST) is positioned dorsolateral of the base of the neural spine of preural 2 (dashed line), dorsolateral of preural 1 and dorsal of urals 1 and 2, reaching posteriorly to a level opposite the anterior edge of hypural 6. The three epurals (E1–3) are positioned dorsal of the dermal bony outgrowth (dashed line) of the stegural. A cartilaginous plate is positioned dorsal of epurals 2 and 3 (*). A small opisthural cartilage (opc) is present at the tip of the notochord. Ventrally, the haemal spine of preural 2 (HS PU 2), the parhypural (PH) and hypural 1 carry proximally a cartilaginous anterior spine (ant S). Posterior of the hypurals (Hy) a fibrocartilaginous plate (cpl) is present. One or two rudimentary hyaline cartilages can be present in the fibrocartilaginous plate.

Figure 3.

Postcranial vertebrae and the caudal fin endoskeleton: key characteristics. (A–G) Parasagittal sections from regions indicated in Fig. 2C. All sections are oriented with dorsal to top (anterior to the left for parasagittal sections). (A–C) Postcranial vertebrae (type I) have an elastic cartilaginous band in the basidorsals (BD, indicated by *), similar to abdominal vertebrae (type II). Ventrally, the recessed parapophysis (arrow) and cartilaginous element are visible in vertebra 1 (V1). The parapophysis on vertebra 2 (V2) is indicated by a black arrowhead as a small sliver of bone in this plane of sectioning. The magnified images in (B) and (C) are indicated in the black boxes. (B) The basidorsal of V2 with the elastic cartilage band indicated by a white *. (C) The basiventral of V2 with the parapophysis indicated by the black arrowhead (A) Masson's Trichrome, (B,C) Verhoeff‐Van Gieson. Scale bars: 100 μm. (D–G) The caudal fin endoskeleton: (D) section through the caudal fin endoskeleton left of the midline of the specimen (see Fig. 2C). Not all structures are visible in this image due to the plane of sectioning. Dorsally, the basidorsals (BD) of preural (PU) 5–4 and ural 1 (U1) are visible. The neural spines of preurals 3 (NS PU 3) and 2, the stegural (ST) and the proximal part of the epurals (E) are visible. Ventrally, the basiventrals (BV) of preural 5–1 and ural 1 are visible. The bony bases of the haemal arches of preurals 5 (arrow) and 4 are fused to the basiventrals. A part of the arch and the distal part of the haemal spine of preural 3 (HS PU 3), the haemal spine of preural 2 (HS PU 2) and the parhypural (PH) are visible. Hypural 1 (Hy 1) and the basal and distal part of hypural 2 (Hy 2) are positioned ventrally of ural 1. Proximally, the anterior spine (ant S) of the preural haemal spines and hypural 1 are sectioned. Distally, the cartilage of hypural 1 and 2 is continuous (*), and the distal part of hypural 3 (Hy 3) is visible. Positioned distally from hypural 2 and 3, rudimentary cartilages (black arrowheads) embedded in the fibrocartilaginous plate can be observed. Haematoxylin and eosin. Scale bar: 200 μm. (E) Fused basiventrals of hypural 1 (Hy 1) and 2 (Hy 2) with each basiventral showing a proliferation zone. Heamatoxylin and eosin. Scale bar: 200 μm. (F) Distally, the cartilage of hypurals 1 and 2 (Hy 1 and 2) are continuous (indicated by *). Masson's Trichrome. Scale bar: 100 μm. (G) Ural 2 (U2) and the bony base of hypural 4 (Hy 4). The bony base of hypural 4 is connected to ural 2 with collagen fibre‐rich tissue and Sharpey's fibres (black arrows). Haematoxylin and eosin. Scale bar: 50 μm.

Figure 4.

Connection of ribs and arches to the abdominal, transitional and caudal vertebral centra. (A–C) Parasagittal sections from the region indicated in Fig. 2B. All sections are oriented with dorsal to the top (anterior to the left for parasagittal sections). (A) Section through an abdominal vertebral centrum and associated elements. Dorsally, the anterior bony outgrowth at the base of the neural arch (arrow), the neural arch itself (NA) and the base of the neural arch (BNA) is visible. The basidorsal (BD) is fused to the base of the neural arch and an elastic cartilaginous band (indicated by *; stained darker) is positioned ventrally within the basidorsal. Ventrally the basiventral (BV) attaches to the mineralised notochord sheath (indicated by arrowhead) of the notochord and is fused to the parapophysis (pap) and distally joined with a rib (R). Scale bar: 100 μm. (B,C) The cartilage of the rib (R) is attached to the parapophysis (pap) with a cartilaginous joint. Elastic/cell‐rich cartilage (white arrow) connects the hyaline cartilage of the rib to the hyaline cartilage of the parapophysis centrally. Fibro/cell‐rich cartilage is positioned closely apposed to the elastic/cell‐rich cartilage, and Sharpey's fibres (black arrows) connect the bone of the rib to the bone of the parapophysis and line the joint. The magnified region in (C) is indicated in the black box. (B,C) Verhoeff‐Van Gieson. Scale bars: 100 μm (B); 50 μm (C). (D,E) Sharpey's fibres (black arrows) connecting the rib (R) and the haemal arch [HA in (E)] in the transitional region (type IIIb) are anchored both in the bone of the rib and the bone of the haemal arch. The section is at the level of the intramembranous bony bridge (indicated by arrowhead) connecting the left and right parts of the haemal arch. The magnified region in (E) is indicated in the black box. (D) Masson's Trichrome, (E) Verhoeff‐Van Gieson. Scale bars: 100 μm (D); 50 μm (E). (F,G) The basidorsals (BD) and basiventrals (BV) in caudal vertebrae are completely surrounded by the cancellous bone (CB) of the vertebral centrum. The base of the left neural arch (NA) and haemal arch (HA) are fused to the cancellous bone of the vertebral centrum. The magnified region in (G) is indicated in the black box. (F,G) Haematoxylin and eosin. Scale bars: 100 μm (F); 50 μm (G).

Table 2.

Definition of the vertebral column regions and types of vertebrae (+ indicates additional structure, > indicates fused structures)

Vertebral type	Definition
Type I	Postcranial vertebrae: vertebral centrum + rudimentary or complete parapophyses + epineurals
Type II	Abdominal vertebrae: vertebral centrum + parapophyses + ribs + epineurals
Type III	Transitional vertebrae: four different subtypes
Type IIIa	Vertebral centrum > open haemal arch (ventrally extended parapophyses) + ribs
Type IIIb	Vertebral centrum > closed haemal arch + ribs
Type IIIc	Vertebral centrum > closed haemal arch > haemal spine + ribs
Type IIId	Vertebral centrum > closed haemal arch > haemal spine + rudimentary ribs
Type IV	Caudal vertebrae: vertebral centrum > neural arch > neural spine > haemal arch > haemal spine
Type V	Preural vertebrae: vertebral centrum + neural arch > modified neural spine + haemal arch > modified haemal spine
Type VI	Ural vertebrae: vertebral centrum + stegural + epurals + uronurals + hypurals

The description below for each of the six regions is based on a typical specimen with 64 vertebral centra.

Postcranial vertebrae (type I)

Postcranial vertebrae (two in total, V1–2) carry epineurals dorsally and parapophyses on the ventral side, and lack ribs (Fig. 1; Table 2).

Epineurals are attached to the base of the neural arches (not shown), and plate‐like pre‐ and post‐zygapophyses are present at the anterior and posterior of the base of the neural arches (indicated by arrows, Fig. 1). The first postcranial vertebra has a ventrolateral vestigial parapophysis consisting of a small cartilaginous element (Fig. 3A) recessed into the cancellous bone of the vertebral centrum. The second postcranial vertebra has a basiventral and parapophysis (Fig. 3B,C) similar to the basiventral‐parapophysis configuration of abdominal vertebrae (Fig. 1). Possible variations include fusion of the first vertebra to the skull (basioccipital) and an additional postcranial vertebral centrum (V3).

Abdominal vertebrae (type II)

Abdominal vertebrae (V3–26) are rib‐bearing and carry epineurals dorsally and parapophyses ventrally (Fig. 1; Table 2).

The rib of the first abdominal vertebra is half the length of the ribs of more posterior abdominal vertebral bodies (Fig. 2A). The abdominal vertebrae carry epineurals attached to the base of the neural arches (not shown). Like in postcranial vertebrae, plate‐like pre‐ and post‐zygapophyses are observed anterior and posterior of the bases of the neural arches (indicated by arrows, Figs 1, 2B and 4A). Ventrally, abdominal vertebrae have basiventrals and parapophyses (Figs 1, 2B and 4A). The cartilaginous part of the rib head is attached to the cartilages of the parapophyses by a cartilaginous joint containing two types of cartilage, elastic/cell‐rich and fibro/cell‐rich cartilage (Figs 2B and 4A–E). The bone adjacent to the cartilage of the rib head is connected to the bone of the parapophysis by collagen fibre‐rich tissue and Sharpey's fibres that envelope the cartilaginous joint (Fig. 4A–C). The number of abdominal vertebrae can vary.

Transitional vertebrae (type III)

The transitional region (V27–36; Figs 1 and 2B; Table 2) shows a stepwise anterior to posterior change from vertebrae with parapophyses and ribs anteriorly, over vertebrae with open haemal arches and ribs, to haemal arch and haemal spine‐bearing vertebrae with vestigial ribs posteriorly. Anteriorly in the transitional region, the bases of the neural arches fuse progressively to the cancellous bone of the vertebral centra (Fig. 2B). Where fusion of the neural arch to the vertebral centrum is complete, the pre‐ and post‐zygapophyses at the base of the neural arch have now a smaller, sharp cone‐like shape (cf. type IV vertebrae, Fig. 2B). The basidorsals gradually reduce in size anterior to posterior in transitional vertebrae. Each transitional vertebral subtype can vary in number. We have therefore distinguished four subtypes.

Subtype IIIa transitional vertebrae (V27–28) have parapophyses and ribs (Fig. 2B; Table 2). Dorsally the base of the neural arch is fusing to the cancellous bone of the vertebral centrum. Ventrally, the anterior part of the parapophysis gradually lengthens and extends ventrally (Fig. 2B; Table 2). In the anterior‐most type IIIa vertebra the head of the rib is attached to the parapophysis with a cartilaginous joint (similar to abdominal vertebrae). In the posterior‐most type IIIa centrum, the cartilaginous joint is lost and the rib is positioned posteroventrally of the parapophysis. The rib remains connected with collagen‐rich tissue and Sharpey's fibres (Fig. 2B).

Subtype IIIb vertebrae (V29–32) have haemal arches and ribs (Fig. 2B; Table 2). Dorsally the base of the neural arch is now completely fused to the cancellous bone of the vertebral centrum. The ventrally extending parapophysis represents an open haemal arch, which increases in length. The base of the open haemal arches fuses to the cancellous bone of the vertebral centrum. The left and right part of the haemal arches fuse distally via an intramembranous bony bridge. The head of each rib remains connected posteriorly with collagen‐rich tissue and Sharpey's fibres (Fig. 4D,E), but gradually lies more ventrally relative to the haemal arches (Fig. 2B). The size of the ribs decreases antero‐posteriorly (Fig. 2A).

Subtype IIIc vertebrae (V33–36) have haemal arches, haemal spines and ribs (Figs 1 and 2B; Table 2). A gradually longer haemal spine is fused to the distal bony bridge of the closed haemal arch. The ribs further decrease in size posteriorly.

Subtype IIId vertebrae (V35–36) have haemal arches, haemal spines and vestigial ribs (Fig. 2B; Table 2). Haemal pre‐ and post‐zygapophyses and a full‐sized haemal arch and spine are present. The vestigial ribs are often positioned more ventrally compared with the ribs in type IIIc vertebrae, and remain connected to the haemal arch with collagen‐rich tissue and Sharpey's fibres (Fig. 2B). Vestigial ribs can have the shape of a miniature rib or be reduced to a small sliver of bone (Fig. 2B). Left–right asymmetry can occur in the shape, size and number of vestigial ribs.

Caudal vertebrae (type IV)

Caudal vertebrae (V37–58) have neural arches and neural spines dorsally, and haemal arches and haemal spines ventrally. Neural and haemal pre‐ and post‐zygapophyses are present (Figs 1 and 2B; Table 2).

The basidorsals and basiventrals of caudal vertebrae are small and completely surrounded by cancellous bone of the vertebral centrum (Figs 2B and 4F,G). The bases of the neural and haemal arches are solidly fused to the cancellous bone of the vertebral centrum. The number of caudal vertebrae can vary.

Preural vertebrae (type V)

Preural vertebrae (V59–62; Figs 1 and 2C; Table 2) carry neural and haemal arches and modified spines to support caudal fin rays (Fig. 2A,C).

Preurals 4‐2 have large basidorsals that are continuous with the cartilaginous neural arches (Figs 1 and 2C). The anterior part of the stegural is positioned directly dorsolateral of the basidorsals of preurals 2 and 1. The basidorsal of preural 1 is not fused to a neural arch (Fig. 2C). Ventrally, the cartilaginous haemal arches of preurals 5‐2 are continuous with large cartilaginous basiventrals. Similarly, the cartilaginous parhypural on preural 1 is continuous with the basiventral, and a semi‐circular proliferation zone is visible (indicated by dashed line, Fig. 2C). The preural haemal spines and the parhypural have anterior and posterior intramembranous bony outgrowths and are interconnected with collagen fibre‐rich tissue and Sharpey's fibres. The haemal spine of preural 2 and the parhypural extend proximally into a perichondrally ossified anterior process. The anterior process has a distinct anterior cartilaginous proliferation zone (indicated by dashed line, Figs 2C and 3D). Distal to the haemal spines of preurals 3‐1 a cartilaginous structure can be present (indicated by *, Fig. 2C). Possible variations include a varying number of preural vertebrae, the fusion of preural vertebrae and the presence (and left–right asymmetry) of a vestigial neural arch on preural 1. The neural arch of the anterior‐most preural vertebra can be fused to the cancellous bone of the vertebral centrum. The cartilaginous structure distally of the haemal spines of preurals 3‐1 can vary in shape and size.

Ural vertebrae (type VI)

The ural vertebrae (V63–64; Table 2) carry the stegural, epurals and uroneurals dorsally and hypurals ventrally. These neural and haemal elements are modified to support caudal fin rays (Fig. 2A,C).

Ural 1, but not ural 2, has a basidorsal (Fig. 2C). The medial and posterior part of the stegural is positioned immediately dorsal of the basidorsal of ural 1 and ural 2, respectively. The medio‐distal intramembranous bony outgrowth of the stegural overlaps with the proximal part of three epurals (Fig. 2C). Distally of epural 2 and 3, a cartilaginous structure is present (indicated by *, Fig. 2C). Uroneural 1 is positioned posteriorly of epural 3 and partly overlaps the dorsal end of the stegural. Uroneural 2 is positioned dorsally of uroneural 1 (Fig. 2C). Ventrally, ural 1 has a single very large basiventral, fused with the basiventrals of preural 1 through cartilaginous protrusions, which show a proliferation zone (indicated by dashed line, Fig. 2C). Within the basiventral of ural 1 itself, two semi‐circular proliferation zones are present (indicated by dashed lines in Figs 2C and 3D, magnified in Fig. 3E). The cartilage of these two zones is continuous with the cartilage of hypural 1 and 2 respectively (Figs 2C and 3D). The bases of hypurals 1 and 2 are both fused to the basiventral of ural 1. Proximally, hypural 1 has a perichondrally ossified cartilaginous anterior process (Figs 2C and 3D). Distally, the cartilage of hypurals 1 and 2 is continuous (Figs 2C and 3D–F). Anterior and posterior intramembranous bony outgrowths are present on hypurals 1–3. The proximal part of hypural 3 is positioned at the level of the intervertebral region of ural 1 and 2. The proximal cartilaginous part of hypural 3 is connected to the proximal cartilaginous part of hypural 2 and the intervertebral region with elastin fibres. The perichondral bone at the base is connected with Sharpey's fibres to ural 2 (Fig. 2C). Ural 2 has no basiventral, and the perichondrally ossified bases of hypurals 4 and 5 are connected with Sharpey's fibres to the bone of ural 2 (Figs 2C and 3G). Hypural 6 is connected with collagen fibre‐rich tissue and Sharpey's fibres to hypural 5 (Fig. 2C). Possible variations include extra ural vertebral centra posterior of ural 2, the absence of epural 3 and/or presence of uroneural 3 and the presence of a seventh hypural. The shapes and sizes of the cartilaginous structure distally of epurals 2–3 can vary. Rudimentary hyaline cell‐rich cartilages (one or two) can be present within the cartilaginous plate (Fig. 2C), and are positioned at the level of the gap between hypural 2 and 3.

Discussion

Irrespective at which of the two temperatures specimens are raised, postcranial, abdominal, transitional, caudal, preural and ural regions are recognised within the vertebral column of Chinook salmon, based on distinct anatomical characters of vertebral centra and their associated elements. The recognition of the transitional region is new as a separate region in‐between the abdominal and caudal region.

If one applies the new system of the recognition of six vertebral column regions to the salmonid vertebral column, in which anomalies frequently occur, most deformities are present in the posterior abdominal (Fjelldal et al. 2007; Sullivan et al. 2007; Fjelldal & Hansen, 2010) and caudal regions (Wargelius et al. 2005; Grini et al. 2011). Although the total number of vertebral centra of specimens in each temperature group is not related to temperature, the number of centra varies in each region. Whether variation in the number of vertebrae is related to temperature is currently being investigated. The transitional region could be more temperature sensitive, for example, showing variation in the number of skeletal elements such as vertebral centra and vestigial ribs related to rearing temperature. The variation of vertebrae within the transitional and other regions is likely related to variation of somite numbers in each region. This is supported by evidence found in threespine stickleback (Gasterosteus aculeatus), where the variation in the caudal, preural and ural regions is due to modulation of somite formation (Lindsey, 1962; Ahn & Gibson, 1999a). Alternatively, a homeotic shift could cause regional specific variation and was suggested as an underlying mechanism in actinopterygians (Ward & Brainerd, 2007; Ward & Mehta, 2010). Either variation of somite number or homeotic shift could cause regional variation in Chinook salmon, but also a synergistic effect of both mechanisms can drive regional variation (Ahn & Gibson, 1999b). In contrast, Ahn & Gibson (1999c) suggest that segmentation and axial regionalisation in teleosts are two separate developmental processes, and this is supported by evidence found in amniotes (Gomez & Pourquié, 2009; Müller et al. 2010). However, in the anterior part of the mammalian vertebral column a strong coupling between the number of vertebrae and the vertebral identity (vertebral column regions) appears evident. Variation of skeletal elements such as vestigial ribs in the transitional region and epurals and uroneurals in the caudal fin could be used as an indicator for developmental stress and stress caused by husbandry practices in fish farming (Leary et al. 1985; Leary & Allendorf, 1989; Parsons, 1992; Allenbach, 2011).

Vertebral centra have been previously recognised as ‘cervical’ in dipnoans and teleosts. Postcranial vertebrae were recognised among others on the basis of lack of ribs in the Dipnoi Neoceratodus forsteri (Queensland lungfish; Johanson et al. 2005), and in the teleosts Gadus morhua (Atlantic cod) and Danio rerio (zebrafish; Morin‐Kensicki et al. 2002; Holley, 2007; Fjelldal et al. 2013). The first postcranial vertebra is known to fuse to the basioccipital in teleosts and is part of natural variation (Johnson & Britz, 2010). Moreover, fusion of vertebral centra to the base of the skull (occipital region) and to the urostyle (fusion of ural vertebrae) is a common non‐pathological phenomenon in osteichthyans, and occurs both in farmed and wild specimens (Arratia & Schultze, 1992; Arratia et al. 2001; Britz & Johnson, 2005; Johanson et al. 2005; Witten et al. 2009). The posterior end of the postcranial region co‐aligns with the anterior expression boundary of the Hoxc‐6 in zebrafish (Burke et al. 1995; Morin‐Kensicki et al. 2002; Holley, 2007). Whether the posterior‐most postcranial centrum coincides with the anterior expression boundary of Hoxc‐6 in Chinook salmon is currently unknown. To our knowledge, transitional centra have never been recognised as a separate region in the literature before. Yet there are clearly recognisable anatomical characters that distinguish transitional vertebrae from abdominal vertebrae and caudal vertebrae, and that support their identification as a separate transitional region: the presence of a haemal arch and ribs on the same vertebral centrum. Although a separate transitional region has never been defined before, vertebrae with transitional morphological characters have been described in basal actinopterygians of the genus Lepidosteus (Balfour & Parker, 1882) and in several teleost species, such as in Atlantic salmon, zebrafish, onion trevally (Carangoides caeruleopinnatus) and striped bass (Morone saxatilis; Götte, 1879; Bird & Mabee, 2003; Holley, 2007; Nowroozi et al. 2012; Jawad, 2015). Transitional vertebrae are distinctive due to the presence and the anatomical shape of the parapophyses. For example, neoscopelid fishes have two series of parapophyses, i.e. a lateral and ventral series (Miyashita & Fujita, 2000). The parapophyses on the anterior transitional vertebrae of the onion trevally are lobe shaped, while long flange‐like parapophyses are present on posterior transitional vertebrae (Jawad, 2015). In frigate tuna (Auxias thazard), the parapophyses on vertebral centra 8–12 have blade‐like shapes with posteriorly bent distal ends (Jawad et al. 2013). Furthermore, parapophyses can have distinctive functions. For example, in black mackerel (Scombrolabrax heterolepis) the parapophyses bulge dorsolaterally to form hemispherical pockets or bullae. These bullae open ventrally to allow evaginations of the gas bladder to fit inside the pockets (Bond & Uyeno, 1981). In Atlantic salmon and zebrafish the parapophyses are connected with a bony bridge to form the haemal canal (Ford, 1937). Parapophyses in the transitional region of Chinook salmon have likely a similar function.

A vertebral column consisting of five regions (cervical, thoracic, lumbar, sacral and caudal) is often considered typical for tetrapods. Interestingly, a tetrapod‐like regionalisation was recognised in the early actinopterygian Tarrasius problematicus and could thus be an ancestral character of osteichthyans (Sallan, 2012). A vertebral column with distinct regions is also present in teleosts; however, the morphology of the vertebral centrum and associated elements likely presents a highly derived state. For example, the transitional region where haemal arches and ribs co‐occur could be a derived character according to Sallan (2012), who discussed that the tetrapod sacral region is anteriorised in teleosts causing ribs and haemal arches to be present on a single vertebral centum. It is tempting to suggest that the ‘lumbar’ and/or ‘sacral’ regions in T. problematicus are comparable to the transitional region described here. Specifically, the position of the first haemal arch (cf. type IIIb vertebrae in Chinook salmon) and co‐occurrence of haemal arches and ribs are common to O. tshawytscha and T. problematicus. Moreover, the stepwise anterior to posterior change of parapophyses into haemal arches is also observed in other actinopterygians and even sarcopterygians (Balfour & Parker, 1882; Nowroozi et al. 2012; Sallan, 2012; Jawad et al. 2013).

Although regions, such as postcranial, abdominal, transitional, caudal, preural and ural, in the actinopterygian vertebral column have been described, the literature is still dominated by the use of a dichotomous subdivision: an anterior and posterior region, often referred to as abdominal and caudal, respectively (Seymour, 1959; Gill & Fisk, 1966; Grande & Bemis, 1998). The anterior region consists of postcranial (type I), abdominal (type II) and transitional vertebrae (type III), and the posterior region of caudal (type IV), preural (type V) and ural vertebrae (type VI). Initially the abdominal‐caudal subdivision of the vertebral column was based on anatomical characters. For example, in the vertebral column of Oncorhynchus spp. the first caudal vertebral body was recognised as the vertebral centrum with a closed haemal arch and median spine as associated elements (Clothier, 1950; Canagaratnam, 1959; Seymour, 1959; Gill & Fisk, 1966). Such a definition would include our transitional region in the anterior region. Recently the abdominal‐caudal subdivision gained support in evolutionary developmental studies, investigating which mechanisms underlie vertebral column subdivision. Ward & Brainerd (2007) hypothesised that the actinopterygian vertebral column contains two axial modules. Several studies on development and evolution of body shape and elongation of fish have collected evidence in favour of a separate evolution of the anterior and posterior axial modules (Ward & Brainerd, 2007; Ward & Mehta, 2010, 2014; Maxwell & Wilson, 2013; Schilling & Long, 2014). For example, most actinopterygian fish evolve elongated bodies mainly by adding vertebral centra rather than by increasing the length of individual vertebrae. The increase in number of vertebral centra occurs either in the abdominal or in the caudal region. Moreover, when vertebral centra are added in the abdominal region little change occurs in the caudal region and vice versa. Ward & Mehta (2010) suggested that a homeotic shift could change the number of vertebral centra in the anterior or posterior vertebral column regions. Such an identity switch could be caused by changes in Hox‐gene expression patterns. In mammalian vertebral columns the Hox‐genes anterior expression boundaries correspond to sharp changes in vertebral morphology. However, the transitional region in Chinook salmon shows a gradual anterior to posterior change of the parapophyses to haemal arches and the regression of ribs to vestigial ribs. The gradual change of structures in the transitional region challenges the correspondence of Hox‐genes expression patterns to sharp morphological boundaries. Only the Hoxc‐6 expression co‐aligns with the postcranial‐abdominal border in zebrafish. In contrast, the Hoxd12a anterior expression boundary is located in the middle of the transitional region and is not co‐aligned with any anatomical boundary (Burke et al. 1995; Morin‐Kensicki et al. 2002; Holley, 2007; Ward & Brainerd, 2007). Except for the Hoxc‐6 anterior expression boundary, there is currently no strong support for co‐alignment of Hox‐gene expression domains and vertebral column regions in actinopterygians. Regionalisation of the anterior part of the actinopterygian vertebral column might be controlled by a different, and currently unknown, mechanism that facilitates homeotic shifts and meristic changes (Ward & Mehta, 2010). Also, a combination of homeotic and meristic mechanisms could underlie axial variation in teleost species. Studies on the threespine stickleback species complex support a synergistic effect of both mechanisms on axial variation (Ahn & Gibson, 1999a,b,c).

Examples of exceptions to the anterior and posterior axial modules suggested by Ward & Brainerd (2007) exist in literature. The position of the anus and the anterior insertion point of the anal fin (Mabee et al. 2002) have been associated with the border of the abdominal and caudal axial modules (Maxwell & Wilson, 2013). However, in a study on the vertebral column of ‘ambush predator’ type teleosts it was suggested that the abdominal/caudal regional border dissociates from the anal fin insertion point. This represents a first example to an exception of the anterior and posterior axial modules. Specifically, the position of the anal fin moves posterior and consequently up to nine haemal arch‐bearing vertebrae are positioned anterior of the anal fin (Maxwell & Wilson, 2013). A second example is represented by the Weberian apparatus in zebrafish (Bird & Mabee, 2003), a modification of the five anterior postcranial vertebral centra, that evolved in the abdominal region. The postcranial vertebral centra and connected elements comprising the Weberian apparatus show marked growth rate differences compared with other vertebral centra in Ostariophysi (Bird & Hernandez, 2009), supporting the idea about the separate evolution of this region of the vertebral column. The dissociation of the position of the anal fin in ‘ambush predators’ and the different growth rate of the Weberian apparatus in Ostariophysi both show that more than two anterior–posterior modules can exist in the vertebral column of teleosts.

Another way of explaining the regionalisation of the vertebral column in teleosts and other vertebrates is to look at centra and associated elements (neural and haemal arches) as separate developmental modules (Holtzer, 1951; Strudel, 1953a,b; Detwiler & Holtzer, 1956; Hall, 1977; Witten et al. 2007; de Azevedo et al. 2012; To et al. 2015). The development of the teleost vertebral centrum starts with the mineralisation of the notochord sheath without the presence of a cartilaginous precursor. Arratia et al. (2001) designates the teleost vertebral centrum anlage as a chordacentrum. Conversely, the arches are somitic (sclerotome) derived and develop from cartilage precursors (Huxley, 1859; Kolliker, 1859; Schaeffer, 1967; Arratia et al. 2001; Grotmol et al. 2003; de Azevedo et al. 2012). Investigation of ‘fused somite’ mutant zebrafish shows that the pattern of neural and haemal arches is disrupted yet the vertebral centra develop normally (Fleming et al. 2004). A recent study on medaka (Oryzias latipes) shows that the ablation of somite‐derived osteoblasts has no influence on the mineralisation of vertebral centra within the notochord sheath (Yu et al. 2017). Furthermore, in farmed Atlantic salmon the neural and haemal arches stay separate when vertebral centra fuse (Witten et al. 2006). These studies show the separation of vertebral centra and associated elements and support the idea that centra and associated elements are developmental modules. Interestingly, also for the Chinook salmon vertebral column the associated elements rather than the vertebral centra themselves are the anatomical structures that determine the regional identity of the centrum and its associated elements.

In conclusion, six anatomical regions, including a new transitional region, represent a refined regionalisation of the vertebral column of juvenile Chinook salmon. The recognition of this refined regionalisation is based on the associated elements of vertebral centra rather than the centra themselves. Studying vertebrate taxa using this refined identification of regionalisation can help to address evolutionary developmental and functional questions related to the gnathostome vertebral column. Understanding of such fundamental questions supports applied research into farmed fish species like Chinook salmon.

Author contributions

A. De Clercq: conceptualisation, methodology, investigation, writing – original draft preparation, and visualisation. M.R. Perrott: conceptualisation, methodology. P.S. Davie: conceptualisation, review and editing of manuscript. M.A. Preece: resources and approval of manuscript (extra term). B. Wybourne: conceptualisation, methodology. N. Ruff: review and editing of manuscript. A. Huysseune: review and editing of manuscript. P.E. Witten: conceptualisation, methodology, review and editing of manuscript.