Functional morphology of the feeding apparatus
Detailed descriptions of the cranial anatomy of Ichthyosaura alpestris and other salamandrids can be found elsewhere [26,32,46–52] and we focus on structures relevant for processing and on specific differences between morphotypes.
Cranial osteology
The feeding apparatus of the Alpine newt consists of an osseous skull and mandible, and a complex, partially cartilaginous hyobranchial system (i.e., hyobranchial in larval or hyolingual in metamorphosed salamanders, respectively) (see Fig. 1) and prominent muscles (Fig. 2). We group the paedomorphic (p) and metamorphic (m) specimens into three distinct morphotypes: (i) late-larval (p), (ii) mid-metamorphic (p), and (iii) post-metamorphic (m) based on their developmental state. The anterior skull plates of the late-larval morphotype (LLM) are largely unfused while in the mid-metamorphic morphotype (MMM) and the post-metamorphic morphotype (PMM) the enlarged frontal bones fill those gaps. The pterygoids of LLM and MMM are relatively small compared to those of the PMM. All morphotypes carry two functional upper jaw systems: the first consists of the tooth bearing maxilla and premaxilla (i.e., primary upper jaw), and the second of the tooth bearing vomerine and palatine bones of the mouth roof (i.e., secondary upper jaw or palatal jaw).
The palatal dentition pattern of the LLM is U-shaped and the teeth organized in rows, the mandible is slightly V-shaped in ventral view and the functional occlusal surface for the lower jaw dentition is the palate between primary and secondary upper jaws. The mandibles of MMM and PMM are U-shaped in ventral view and the occlusal surface for the lower jaw are the maxillary teeth of the primary upper jaw. The palatal dentition of the MMM and the PMM are distinct as the MMM has a U‑shaped single row of denticles and the PMM exhibits a V‑shaped single row of denticles.
Hyobranchial musculoskeletal anatomy
The hyobranchial apparatus shows the most striking differences between morphotypes. In the LLM, the hyobranchial apparatus is a complex and mainly cartilaginous system with small ossification centers in ceratohyal, hypobranchial and urohyal. The hyobranchial apparatus of MMM shows enlargement of these ossified centers, additional ossification centers in basibranchial, ceratobranchial 1 and 3, as well as the reduction of the urohyal. The hyolingual apparatus of the PMM exhibits a typical morphology for metamorphosed salamandrids. Thus, in the PMM the ceratobranchial 2 - 4 are reduced and the hypohyals merge to form a buckle around basibranchial (often referred to as the radial).
Our functional descriptions of the hyobranchial apparatus focus on muscles responsible for the main movements of the anterior tip of the hyobranchial system (i.e., the basibranchial). The 3D muscle morphology is considered but the main function of each muscle is assessed from a lateral perspective (i.e., simplified to a 2D movement). More complex inter-hyobranchial movements are likely to occur due to the 3D orientation of the hyobranchial apparatus and its muscles (see for example [53]). The hyobranchial system of all morphotypes forms the attachment site for several major muscles (i.e., six in the LLM and MMM, and five in the PMM). The muscles can be differentiated according to their initial attachment to hyoid arch or branchial arch during ontogeny [54]. The hyoid arch (paired ceratohyals) is connected with the ceratomandibularis (CM) and branchiohyoideus externus (BHE) in all morphotypes, and also with the levator hyoideus (LH) in the LLM and MMM. The CM extends between the ossified area of the ceratohyal and the dentary in all morphotypes, acting as a protractor of the hyobranchial system. The fleshy BHE extends from the lateral side of the postero-dorsal ceratobranchial I to a tendoninous sheet connecting the anterior regions of the hyoid- and branchial arch in the LLM. Because of the ligamentous connection of the ceratobranchial to the mandible (HML), the BHE serves as a retractor of the anterior part of the branchial system while it adduces the anterior tip of the hyobranchial apparatus and the posterior part of ceratobranchial I. In contrast, in the MMM and the PMM, the insertion of the BHE shifted completely to the antero-ventral part of ceratohyal and therefore acts as a protractor of the branchial arch. In the LLM, the LH originates on the dorsal squamosal process and attaches to the upper osseous part of the ceratohyal, while in the MMM the LH originates from the mid-squamosal and attaches to the upper osseous part of ceratohyal. Accordingly, the LH serves as a hyobranchial elevator in LLM and MMM. The LH is missing in the PMM because the LH detaches from the hyobranchial system during development in order to attach to the lower jaw and thus form the depressor mandibulae posterior. Apart from the development of the depressor mandibulae posterior, the cranial muscles for opening and closing the jaw showed no significant differences between the morphotypes.
The branchial arch is connected with geniohyoid (GH), subarcualis rectus 1 (SR1), and rectus cervicis (RC). The thin GH muscle extends from the basibranchial to the dentary in all morphotypes, thus enabling protraction of the hyobranchial system. A peculiarity in metamorphs is that some fibers of the GH extend from the pericardium to the dentary [47]. In the LLM, the SR1 extends from the antero-ventral side of the cartilaginous ceratobranchial I anteriorly to a tendoninous sheet connecting the anterior regions of the hyoid- and branchial arch. Thus, the SR1 acts similarly as BHE in the LLM, retracting the tip of the hyobranchial system, while in the MMM and the PMM the SR1 extends from the medial part of the ceratobranchial I to the medio-lateral part of the ceratohyal to act as a protractor of the branchial arch. The most prominent muscle of the hyobranchial system in all morphotypes is the RC that originates from the ventral abdominal trunk muscles and inserts onto the basibranchial. Due to its course and the ligament and muscle suspension of the hyobranchial apparatus on the skull (hyomandibular or hyoquadrate ligament and levator hyoideus), the RC facilitates retraction and depression of the hyobranchial apparatus.
Intraoral food processing
After initial ingestion via suction feeding, one or two transport movements were used by all morphs to position prey prior to a consecutive set of processing cycles. The mean total processing cycles were 5.7 ± 3.2 (mean ± S.D.) for the late-larval, 5.6 ± 2.4 for the mid-metamorphic, and 5.9 ± 2.5 for the post-metamorphic morphotypes. A processing cycle was defined from start of gape opening until the next start of gape opening. Processing involved the cyclical opening and closing of the jaw (i.e., arcuate mandible movement), elevation and depression of the hyobranchial apparatus (i.e., the tongue) and, in the post-metamorphic morphotype only, additional rhythmic flexion and extension of the neck (vertical cranial movement) (Fig. 3 C). During these movements, prey debris and haemolymph were occasionally expelled from the oral cavity, indicating that the behavior caused significant prey disintegration. After a processing bout (i.e., a series of processing cycles), water flows induced by hyobranchial movement transported the food backwards, after which it was either repeatedly processed or swallowed.
Kinematics of intraoral food processing
Intraoral food processing cycles were clearly distinguishable from food transport in that hyobranchial elevation accompanied gape opening during processing, whereas during transport hyobranchial depression accompanied gape opening. During processing, at the onset of gape opening, the LLM initiated hyobranchial elevation, which continued past peak gape opening and reached its peak coincident with complete gape closure. Then, in a returning motion, the hyobranchial apparatus was depressed while the mouth remained shut (i.e., stationary phase). The MMM started elevating the hyobranchial apparatus at the onset of gape opening. Both movements peaked approximately at the same time, after which simultaneous gape closing and hyobranchial depression (i.e., resetting movements) occurred (Fig. 4 B and H). Neither the LLM nor the MMM had stereotypic cranial movements, as indicated by their relative featureless cranial kinematic profiles (Fig. 4 D, E). In the PMM gape and vertical cranial flexion peaks were approximately coincident; thus, gape opening and cranial ventroflexion (or head depression) as well as gape closing and cranial dorsoflexion were aligned (Fig. 4 C and F). The vertical hyobranchial movement had a ~10% phase shift (i.e., delay) from the gape cycle as hyobranchial elevation started at ~90% of the preceding gape cycle (compare Fig. 4 C and I).
Table 1 shows the kinematic parameters of food processing in the three morphotypes. The stationary gape phase in the LLM clearly differed from the other two morphotypes (compare Fig. 4A with B and C) as did the cranial flexion of the PMM (compare Fig. 4F with D and E).
Table 1: Kinematic parameters of intraoral food processing of three morphotypes of I. alpestris
Kinematic variable
|
Kinematic parameter
|
Late-larval morphotype (LLM)
|
Mid-metamorphic morphotype (MMM)
|
Post-metamorphic morphotype (PMM)
|
Mean ± S.D.
|
CV
|
Mean ± S.D.
|
CV
|
Mean ± S.D.
|
CV
|
Gape
|
1 Opening (°)
|
5.35±1.54
|
0.29
|
9.82±4.28
|
0.44
|
19.30±5.90
|
0.24
|
2 Closure (°)
|
5.51±1.21
|
0.22
|
9.48±4.68
|
0.49
|
19.66±5.86
|
0.23
|
3 Opening duration (s)
|
0.04±0.02
|
0.45
|
0.13±0.06
|
0.48
|
0.16±0.06
|
0.32
|
4 Closure duration (s)
|
0.06±0.02
|
0.36
|
0.11±0.04
|
0.38
|
0.12±0.06
|
0.44
|
5 Closure acceleration (10-3 deg/s²)
|
21.40±6.15
|
0.29
|
12.25±6.99
|
0.57
|
18.75±10.47
|
0.45
|
6 Open-close duration (s)
|
0.10±0.03
|
0.25
|
0.24±0.07
|
0.27
|
0.28±0.07
|
0.21
|
7 stationary duration (s)
|
0.17±0.08
|
0.45
|
n/a
|
n/a
|
n/a
|
n/a
|
8 Cycle duration (s)
|
0.28±0.09
|
0.33
|
0.24±0.07
|
0.27
|
0.28±0.07
|
0.21
|
Vertical cranial flexion
|
9 Ventral (°)
|
n/a
|
n/a
|
n/a
|
n/a
|
12.61±6.51
|
0.39
|
10 Dorsal (°)
|
n/a
|
n/a
|
n/a
|
n/a
|
12.30±6.46
|
0.40
|
11 Ventral duration (s)
|
n/a
|
n/a
|
n/a
|
n/a
|
0.10±0.05
|
0.41
|
12 Dorsal duration (s)
|
n/a
|
n/a
|
n/a
|
n/a
|
0.17±0.07
|
0.33
|
13 Cycle duration (s)
|
n/a
|
n/a
|
n/a
|
n/a
|
0.28±0.07
|
0.21
|
Vertical hyobranchial movement
|
14 Elevation (%-cl)
|
4.29±1.99
|
0.46
|
4.93±1.47
|
0.30
|
10.57±6.18
|
0.53
|
15 Depression (%-cl)
|
3.87±1.34
|
0.35
|
5.43±1.50
|
0.28
|
12.46±5.64
|
0.36
|
16 Elevation duration (s)
|
0.08±0.02
|
0.24
|
0.14±0.06
|
0.45
|
0.13±0.05
|
0.34
|
17 Depression duration (s)
|
0.20±0.09
|
0.44
|
0.10±0.03
|
0.35
|
0.14±0.05
|
0.31
|
18 Cycle duration (s)
|
0.28±0.10
|
0.35
|
0.23±0.07
|
0.28
|
0.27±0.07
|
0.23
|
Abbreviations: S.D. standard deviation, CV coefficient of variation and n/a not applicable. Note that parameters 6 and 8 are identical for both MMM and the PMM. This is because both MMM and PMM lack a stationary phase during processing (parameter 6), so that opening and closing the mouth corresponds to the gape cycle. Note the stereotypy of the magnitude of gape movements (parameter 1 and 2) in the LLM, the flexibility of the gape movements (parameter 1-4) in the MMM, the stereotypy of the hyobranchial movements (parameter 13,14, and 17) in the MMM, the stereotypy of the gape movements (parameter 1,2,3, and 5) in the PMM, and the flexibility of hyobranchial movements (parameter 13-15) in the PMM.
Table 2: Statistical analysis of intraoral food processing kinematics in I. alpestris
Kinematic variable
|
Kinematic parameter
|
All Morphotypes
|
LLM vs. MMM
|
LLM vs. PMM
|
MMM vs. PMM
|
Kruskal-Wallis H
|
p-value
|
Mann-Whitney U
|
p-value
|
Mann-Whitney U
|
p-value
|
Mann-Whitney U
|
p-value
|
Gape
|
Opening
|
78.08
|
0.00*
|
-21.42
|
0.32
|
-80.09
|
0.00*
|
-58.67
|
0.00*
|
Closure
|
79.09
|
0.00*
|
-17.58
|
0.55
|
-78.61
|
0.00*
|
-61.03
|
0.00*
|
Opening duration
|
44.82
|
0.00*
|
-57.24
|
0.00*
|
-73.85
|
0.00*
|
-16.61
|
0.23
|
Closure duration
|
30.18
|
0.00*
|
60.29
|
0.00*
|
59.85
|
0.00*
|
-0.43
|
1.00
|
Closure acceleration
|
17.15
|
0.00*
|
51.24
|
0.00*
|
22.12
|
0.12
|
-29.12
|
0.00*
|
Open-close duration
|
48.69
|
0.00*
|
-61.43
|
0.00*
|
-77.14
|
0.00*
|
-15.72
|
0.28
|
Cycle duration
|
3.56
|
1.00
|
n/a
|
n/a
|
n/a
|
n/a
|
n/a
|
n/a
|
Vertical hyobranchial movement
|
Elevation
|
32.10
|
0.00*
|
-10.04
|
1.00
|
-49.48
|
0.00*
|
-39.45
|
0.00*
|
Depression
|
64.27
|
0.00*
|
-18.06
|
0.52
|
-71.98
|
0.00*
|
-53.92
|
0.00*
|
Elevation duration
|
18.37
|
0.00*
|
-49.55
|
0.00*
|
-45.79
|
0.00*
|
3.76
|
1.00
|
Depression duration
|
33.78
|
0.00*
|
70.21
|
0.00*
|
23.89
|
0.09
|
-46.32
|
0.00*
|
Cycle duration
|
5.22
|
0.88
|
n/a
|
n/a
|
n/a
|
n/a
|
n/a
|
n/a
|
Statistical analysis was calculated using Kruskal-Wallis 1-way ANOVA and only performed on parameters present in all morphotypes. P-values were Bonferroni adjusted to account for multiple testing; significant p-values are indicated by asterisks.
Some significant changes concern the duplication of the vertical hyobranchial magnitude of the PMM compared to the MMM (compare with Fig. 4H and I), the duplication in gape magnitude from the MMM to PMM (compare Fig. 4B and C), and the significantly higher mean mandible acceleration from peak gape opening to reaching maximal gape-closing speed in the LLM compared to the MMM. The durations of the gape and vertical hyobranchial movement cycle are the same across all morphotypes.
Ordination analysis of processing kinematics
A principal component analysis (PCA) was performed to analyze how the processing kinematics of the three morphotypes relate to each other and to visualize differences. Distribution of the chewing cycles among the processing modes and morphotypes on the first two principal components axes are shown in Figure 5, and the loadings of the kinematic parameters on principal component 1 and 2 (i.e., PC1 and PC2) are given in Table 3. Hyobranchial kinematics load more strongly on PC1 while mandible kinematics loaded more strongly on PC2. Processing in PMM and LLM are separated in kinematic space with no overlap, but MMM processing overlaps with both LLM and PMM.
The coefficient of variation (CV) was calculated for each kinematic parameter (Table 1) in order to quantify the stereotypy of the processing behavior of each morphotype [55]. The stationary gape phase (i.e., parameter 6) was only part of the processing mechanism in the LLM and parameters concerning vertical cranial flexion (8-12) could only be analyzed for the PMM. Consequently, these parameters were excluded for comparison.
Table 3: Loadings of processing parameters on the first two principal components (PC1 and PC2)
Parameter
|
PC1
|
PC2
|
(18) Duration hyobranchial movement cycle
|
0.836*
|
-0.123
|
(15) Magnitude hyobranchial depression
|
0.779*
|
0.353
|
(14) Magnitude hyobranchial elevation
|
0.663*
|
0.480
|
(1) Magnitude gape opening
|
-0.036
|
0.872*
|
(3) Duration gape opening
|
0.300
|
0.699*
|
Total variance explained (%)
|
48.1
|
21.0
|
Parameters marked with * load strongly (> 0.5) on each respective principal component. Note that parameters connected to hyobranchial movement load more strongly in PC1 while parameters connected to gape movements load more strongly on PC2.
Stomach content analysis
Post-metamorphic newts used in stomach content analysis applied suction feeding to ingest lake fly larvae (Chironomidae). After ingestion, the newts used cyclic processing movements involving ventral cranial flexion and mouth opening accompanied by hyolingual elevation. Microscopic examinations of the processed lake fly larvae extracted from the stomachs of freshly euthanized newt specimens revealed clear lesions and other structural damage. Lesions were recognized by intensified methylene blue staining, which gradually attenuated along the unharmed part of the prey (Fig. 6B - D). By contrast, unprocessed lake fly larvae (control) only showed blue coloration in the posterior most region (Fig. 6A) and no structural damage. From a total of 100 processed lake fly larvae, 61 exhibited minor to major structural damage (Fig. 6B - C), 18 were ruptured (Fig. 1 D) and 21 did not show evidence of damage (Fig. 6A).