Methods and material
We employed 43 characters to analyze patterns of relationships throughout the Ingolfiellidea. Some 30 characters are multistate, while 13 features are binary. Following here is a list of the characters employed and an explanation of the several alternative states we have used in constructing a matrix (Table I). Given the ‘reduced’, worm-like body plan of these animals our 43 characters essentially covered all aspects of the recognizable anatomy, so there has been no emphasis of one aspect of morphology over another. The characters we used are as follows.
Character matrix. (Part 1.) The top row of figures (1-43) represents the characters used in a parsimony analysis in PAUP 4.0 b10 (Altivec). Multiple scores in one cell refer to multiple states present in one character.
Character matrix. (Part 2.) The top row of figures (1-43) represents the characters used in a parsimony analysis in PAUP 4.0 b10 (Altivec). Multiple scores in one cell refer to multiple states present in one character.
1. Ocular (cephalic) lobes
state 0 = developed
state 1 = reduced
state 2 = absent
In one of the out-group taxa chosen, Mictocaris halope, this character is termed the eyestalk. Its location is not between antenna 1 and antenna 2 on the front margin of the cephalon, as in the Ingolfiellidea, but is rather located a little higher, flanking the peduncle of antenna 1. Still we consider this possible remnant of a stalk in Mictocaris as an homologous feature with the typical ingolfiellidean cephalic lobe, simply because no other function can be ascribed with certainty to this lobe at the moment. Lowry and Poore (1989) observed that three peracaridan orders have representatives with eyestalks or remnants of stalks, i.e., the mysidaceans, the spelaeogriphaceans, and the mictaceans. However, they conclude that the typical position of the reduced stalks, which are often in the form of scales or pointed lobes, lies at the base of the first antenna and at the rostrum. This is different from that seen in the Ingolfiellidea, where, as noted, the lobes reside between the first and second antennae. Therefore, they do not regard these features as homologous and thus not of subordinal importance.
However, spelaeogriphaceans and mictaceans do not have the lateral compressed head of ingolfiellideans (and of most amphipods) and the position of the lobes could easily have shifted ventrally when that would be the case. We have made SEM photographs of two ingolfiellid species, Ingolfiella ischitana (Fig. 2a) and I. putealis (Fig. 3a,b), in which the difference between the developed state of the lobe in the former (Fig. 2, b-e) and the reduced state (Fig. 3, a-c, e, f) in the latter is clearly visible. In both cases, there is a neat fit of this lobe between the bases of the first and second antennae, and this suggests that the original function (eye-stalk) has been replaced by a new function. The lobes close off the otherwise open space between the antennae to mud particles. Thus the appressed lobes and the rostrum form a tight seal around the protruding antennal peduncles, preventing fine granular material from fouling the head region.
2. Antenna 1, flagellum
state 0 = longer than basal peduncular segment
state 1 = medium length, more than half of basal peduncular segment
state 2 = short, less than half of basal peduncular segment
The low number of segments (4) on the antennal flagellum is a consistent character throughout the Ingolfiellidae and as such not informative towards distinguishing between species or species groups within the family. However, the length of the flagellum as a whole does vary in a few cases, and the differences were scored. Out-groups have more segments, as is common in most peracaridans, and thus bear a relatively long antennal flagellum (state 0). Absolute measurements of the flagellum depend on the size of the individual. With a ratio character like this it is often difficult to divide continuous distributions into integral states. Still an effort is made to distinguish between longer and shorter antennae as our goal was to score as much character differentiation as possible.
3. Antenna 1, accessory flagellum
state 0 = four segments
state 1 = three segments
state 2 = two segments
The accessory flagellum is positioned on the inner side of the flagellum (Fig. 3d, 5k 7c) and could be called the “inner” flagellum, if we want to keep the terminology that would be comparable to early, fossil finds of diverse crustaceans. The positional homology is important here for in Mictocaris the outer flagellum has eight segments and the inner has four segments. We might ask if the accessory flagellum in amphipods is comparable to the outer or inner flagellum of Mictocaris?
“The primitive biramous origin is retained in the form of a small accessory flagellum that arises from the end of the peduncle,” (Lincoln, 1979, p.16). In Mictocaris, the eight-segmented “outer” flagellum bears the aesthetascs, which would suggest the four-segmented “inner” flagellum, which lacks aesthetascs, is equivalent to the accessory flagella in amphipods, also lacking aesthetascs.
Fig. 3. Ingolfiella putealis, paratype. SEM photographs of: a – c, e,f, views from different angles on a small cephalic lobe. Photo c reflects best the possible ‘closing off’ function of the lobe, a similar fit is made by the tiny rostrum (d).
4. Mandibular palp
state 0 = present
state 1 = vestigial
state 2 = absent
The mandibular palp is lacking in the Ingolfiellidea. This implies that the palp is not necessary towards either securing and moving larger food particles to the mouth and the molars, or in cleaning and grooming, as is typically the case for the larger palps seen in malacostracan crustaceans. Fine food particles make up the diet in this line of reasoning. One genus in the Ingolfiellidae, Stygobarnardia Ruffo 1985, possesses a rudiment of the mandibular palp and is scored as vestigial (1).
This reduction and/or complete loss of a mandibular palp occurred in many groups of crustaceans (Richter and Scholtz, 2001) and shows a great variability within families and genera of Amphipoda. For instance, a mandibular palp may be present or absent within the Ampithoidae and Hadziidae, or it may vary in length as in Metacrangonyx. Even so, this phylogenetically uninformative character is retained here to emphasize the special situation in Stygobarnardia and to be available for use with possible finds of new taxa.
5. Molar process size
state 0 = well-developed lobe
state 1 = vestigial peg or spine
All Ingolfiellidea share the reduced state (Figs. 5c, 6l, n, arrows). In combination with the absence of mandibular palps, this feature perhaps points to a lack of need for structures to chew up large particles of food.
state 0 = numerous
state 1 = four or more
state 2 = three
state 3 = two
state 4 = one
state 5 = none
This character is constant within those taxa wherein populations were used for species descriptions, thus it seems significant at least for differentiation of species. This feature is often mentioned only in the ‘remarks’ section of species descriptions as one that differs between species. The condition in which the lobe is fringed with numerous hairs seems to point to an original state. Decidedly more generalized forms, such as Euphausiacea, have many setae on the inner lobe of the maxillule (Maas & Waloszek, 2001)
state 0 = simple
state 1 = bifid
state 2 = dentate
When comparing the tips of the robust apical setae on the distal margin of the outer lobe of maxilla 1, clear differences were noticed between several species. In most species the inner seta stands apart from the others and is implanted on the lateral/submarginal, inner side of the plate. Often it is clearly dentate with a little comb, at other times it is simple.
state 0 = simple
state 1 = bifid
state 2 = dentate
“Simple” setae are smooth, spiniform setae without hooks. In case more states are present, a multistate score is used (0/1 or 0/1/2). Comparison with the out-group Mictocaris halope seems to indicate that an earlier condition might have resembled an undifferentiated tuft of soft setae (Bowman & Iliffe, 1985)
9. Maxilla 1, outer lobe, setae orientation
state 0 = continuous row
state 1 = 7 + additional inner seta
state 2 = 7
state 3 = 6 + additional inner seta
state 4 = 6 (Fig. 5a)
state 5 = 5
The setae on the distal margin of the outer plate of the maxilla 1 can differ in number. The additional seta often sits a little sideways on the inner margin of the lobe. We have no preconceived notion as to what might be the derived situation. We suspect, however, that the most reduced situation (5 setae) reflects the derived state here.
10. Maxilla 1, palp size
state 0 = larger than outer lobe
state 1 = subequal to outer lobe
state 2 = smaller than outer lobe
The need for elaborate handling of larger food items may require a long palp (0) as opposed to a short palp (2). Assumption of the interstitial mode of life, generally supposed to be a secondary habitat choice in the evolution of Amphipoda, may have induced the loss of importance for a maxillular palp.
11. Maxilla 1, palp setae
state 0 = four setae or more
state 1 = three (Fig. 6j, m)
state 2 = two
state 3 = one
state 4 = absent
The number of palp setae is a constant feature in those cases where larger numbers of individual specimens of a species where studied by us. Therefore, it is a useful diagnostic character. A decrease in the number of these setae is noted for what is seen in the out-group taxa.
12. Maxilla 2, setae number outer lobe
state 0 = large complex limb with many setae
state 1 = five setae, or more
state 2 = four setae (Fig. 6k)
state 3 = three setae
state 4 = two setae
The maxilla 2 is a greatly reduced limb with little, aside from setal number, to distinguish variation.
13. Maxilliped basis
state 0 = free and separate
state 1 = fused base proximally
Free and separate bases of the maxilliped suggests a condition close to the plesiomorph situation, e.g., (0) where the maxillipeds are a pair of thoracic walking limbs. The basis is free and separate in mictaceans and in Metaingolfiella (Ruffo, 1968, fig II, 3)
14. Maxilliped lobes
state 0 = with basal lobe only
state 1 = with basal and ischial lobes
Ischial lobes on the maxilliped are lacking in Ingolfiellidea and in Mictacea (0). Other members of the out-groups we employed, the bogidiellids and the pseudoingolfiellids, do have ischial lobes (1). So this character adds no information to in-group polarization of character states. It merely shows the convergent development of pseudoingolfiellids as compared to Ingolfiellidea.
15. Maxilliped, medial palp setation
state 0 = numerous
state 1 = 1 or 2 per segment
Only Metaingolfiella and the other members of the out-group share the primitive state wherein the setation displays the ‘numerous’ condition (0). The Ingolfiellidae all share the derived condition of a reduction to 1 or 2 setae per palp segment (1).
16. Maxilliped, lateral propodal setae
state 0 = present
state 1 = absent
The presence of this row of setae is recorded in only a few instances.
17. Maxilliped, dactyl claw
state 0 = absent
state 1 = single setae
state 2 = robust spine
state 3 = spine with flanking setae
state 4 = falcate (Fig. 5e)
There is considerable variety in the form and number of setae on the apex of the maxilliped palp. No apparent difference in function can be ascribed to forms with one or more spines at the apex. One might be tempted to speculate that blunt and strong spines may assist in heavy food particle holding. However, without functional studies of live material such speculations are of only anecdotal interest.
18. Second thoracic segment (= first pereionite)
state 0 = free
state 1 = fused to cephalon
The situation in the family Metaingolfiellidae is different from that seen in all other species in the Ingolfiellidea and in the chosen members of the out-groups. Although the half-fusion of the cephalon is an autapomorphy and thus phylogenetically uninformative in this analysis, this character cannot be ignored and deserves a place in the matrix to emphasise its peculiarity. Another type of partial fusion of the cephalon and first pereionite can be observed in Caprogammarus gurjanovae Kudrjaschov & Vassilenko, 1966 (in Takeuchi & Ishimaru, 1991). This particular member of the suborder Caprellidea also exhibits a partial fusion of the cephalon and first pereionite but on the ventral side of the segment, while in Metaingolfiella the fusion starts from the dorsal side (Figs. 4a-e). However, the position of the suture is similar in the two genera, and corresponds with the position of first gnathopods, which are shifted anteriorly from their more typical location more posteriad.
19. Pereional segments
state 0 = deeper than long
state 1 = subrectangular
state 2 = elongate
This character serves to capture the overall body habitus (Fig. 2a). This may be an important feature in regard to the habitat requirements. Life in finer sand interstices calls for a “worm-like” appearance.
20. Coxal plates
state 0 = not developed, or weakly so
state 1 = robust
Part of the out-group, but not Mictocaris, exhibits the robust development of the coxal plates (1) more typical of amphipods as a whole. It is a unique character of the Ingolfiellidea that the coxal plates are rudimentary (0). For the Ingolfiellidea as such it is an uninformative state. Coxal plates may have arisen separately on different occasions. For instance, they are present in both amphipods and isopods but form in this case one of the few specific characters common to both groups (Siewing, 1963: p.96)
21. Lenticular organs
state 0 = absent
state 1 = incipient
state 2 = well developed
This is a feature encountered, within Ingolfiellidea, only in species of the African trogloleleupians. The lenticular organs are distinctive, semi-transparent, “windows” of cuticle on the side of the segments of the pereion and pleon (Griffiths, 1989). Their function is unknown. They resemble in some respects the foramen ovale, a similar transparent area of cuticle on the heads of the syncarid genus Allanaspides – another meiofaunal crustacean. In Pseudoingolfiella there is mention of “hyaline spots” but in a different location. Noodt (1959, p. 203) remarks on Pseudingolfiella chilensis: “todos los segmentos toracicos libres poseen entre la insercion de las extremidades un punto circular hialino bien delimitado de funciÒn desconocida”.
state 0 = simpe limb
state 1 = subchelate
state 2 = carpo-subchelate
Since all ingolfiellideans have the carpo-subchelate state (2) this character gives no information of generic or specific relationships within the suborder. The feature is not unique either because, as is remarked by Lowry & Poore (1989), it is also seen in relatively unrelated groups such as the the Pardaliscidae, Aoridae, Corophiidae, Leucothoidae, and the Hyperiidea. Ischyroceridae have also carpochelate second gnathopods (Lowry & Springthorpe, 2001) Nevertheless, this feature is scored here because the out-group does show states 0 and 1.
23. Gnathopod 1, dactyl
state 0 = no gnathopod
state 1 = simple
state 2 = serrate
state 3 = blade-like
state 4 = spines
The simple state is reflected in a smooth posterior margin of the dactylus. This is encountered in Metaingolfiella and Stygobarnardia but also in Ingolfiella littoralis (Fig. 6a). The serrate state (Fig. 7a) is exemplified by I. abyssi. Blade-like forms are observed in two species of Trogloleleupia and in Ingolfiella britannica. Here the spines on the posterior margin are broad and flattenend or, another way of interpreting, the serrated margin has become more widely interspaced. An intermediate form of this is shown in Fig. 5h, on the gnathopod 2 of Trogloleleupia leleupi. When the serrations are broad in contrast to the space between, these would be interpreted as blades. The fourth state, spines, is scored for species with clearly distinguishable rounded spines protruding from the margin.
24. Gnathopod 2 carpus, palmar angle spines
state 0 = unspecialized
state 1 = with elongate spine, not angulate
state 2 = pedicillate
state 3 = protruding angulate process, with spine
The palmar angle spines are often used in amphipod taxonomy as an important character in helping determining differences between species within one genus. These spines are often quite robust and placed at the end of where the tip of the dactylus reaches the inner margin of the propodus/carpus. In some cases the palm makes an angle here as in Trogloleleupia leleupi (state 3, angulate, Fig. 5h). When this seta is placed on a small distinct pedestal we call this pedicillate (state 2, not illustrated). In the case of a straight palmar margin line and a relatively long seta this is scored elongate (state 1, Fig. 6b), and in the unspecialized state (0) the spine is relatively short (Fig. 7b) or not present.
25. Gnathopod 2, carpal saw
state 0 = absent
state 1 = serrate (Fig. 6b)
state 2 = dulled (Fig. 5h)
state 3 = setose brush
state 4 = finely serrate
We found this feature to be very consistent in a sizeable population example in at least one population of Ingolfiella canariensis. We examined more than 50 individuals collected from beach interstitia on the Canary Islands, and all specimens exhibited the serrate state (1) (Vonk & Sánchez, 1991).
26. Gnathopod 2, distal propodus form
state 0 = unmodified
state 1 = tooth-like
state 2 = finger-like
state 3 = blade-like
The propodus in Ingolfiella is typically smaller than the carpus and is positioned at a place where normally the dactylus is attached, i.e., just distal to the sub-flexure. This article displays a spur-like process at its distal end that varies in form from pointed, to blunt, to blade-like, to unmodified. The propodus in Ingolfiella littoralis shows the unmodified state (Fig. 6b, arrow), Trogloleleupia leleupi has the finger-like condition (Fig. 5h, arrow) and Ingolfiella abyssi the tooth-like (Fig. 7b, arrow).
27. Gnathopod 2, dactyl teeth
0 = absent
1 = 3 teeth (Fig. 6b)
2 = 3 blades (Fig. 5h)
3 = 4 teeth
4 = 4 blades (Fig. 7b)
We observed differences in the form of the teeth lining the inner margin of the dactyl of the gnathopods. These thorn-like structures probably help in securing prey and/or mates, or to strengthen the grip when the claspers are used to pull the animal forward. In some species, these teeth are broad and formed into blades. In other species, these structures are more rounded and form tooth-like outgrowths of the cuticle. The number of the structures varies and is also incorporated as a character state.
28. Gnathopod 2, dactyl tip
0 = simple (thin moderate)
1 = simple and thick
2 = long and thin
3 = long and thick
The tip of the dactyl could be perceived as a combination of a proximal “dactylar” part and the distal “ungular” part, although a suture is not easily observed. Some species, like Ingolfiella canariensis and I. similis, have a visible division (Fig. 7b); other species do not. The length and thickness of this tip is variable and can be scored.
29. Gnathopod 2, size
0 = subequal to P3
1 = larger
2 = smaller
The second gnathopod is a powerful tool for an ingolfiellid. It is more robust than the first gnathopod, which has a carpus that is often less broad and more pointed (Figs. 7a, b; 8a, b). The size of the second gnathopod relative to the third pereiopod is measured by summing the lengths of the five podomeres of both appendages.
30. Gnathopod 2, palm
0 = transverse
1 = oblique
The form of the carpus is either robustly triangular with a short palmar margin, “transversely” cut when seen from the side, as in I. littoralis (Fig. 6b), or elongate with a faint sloping, oblique palmar side as in I. abyssi (Fig. 7b).
31. Pereiopods 3 and 4, claw
0 = absent
1 = simple
2 = dentate or bifid
Spiny structures on the termini of the third and fourth pereiopod may be used for better grip. Most species in Ingolfiella have the dentate or bifid state (Fig. 7d), and only a few possess a simple spine. All species in Trogloleleupia, the large African cave species, have the simple state.
32. Pereiopods 3 to 7, dactyl form
0 = similar
1 = dissimilar
A common feature in gammaridean amphipods involves the different positioning of the third and fourth pereiopod in relation to other pereiopods, e.g., the fifth, the sixth, and the seventh. Pereiopods 1 to 4 form an ‘embryological unit’ (Dahl, 1977), or, as we would interpret, a functional/morphological unit. However, the form of the dactylus also can differ between those of P3 and P4 on the one hand, and those of P5-P7 on the other. For instance in I. littoralis the forms of the dactylus on P3 and P4 (Fig. 6c, d) are dissimilar (state 1) from P5 (Fig. 6e).
33. Pereiopods 5-7, dactyli
0 = with claw
1 = without claw
34. Pereiopods 3-7, dactyl ends or termini
0 = not produced
1 = with spur (Fig. 6c)
2 = with seta (Fig. 7d)
This character involves an added bit of decoration to the distal aspect of the dactyli. The spur is a robust spike-like process and is opposed to a more flexible setose extension.
35. Pleopods 1 – 3, female
0 = present
1 = absent
There is a trend in ingolfiellids toward reducing the pleopods to a minimum number. The absence of all the pleopods would be the ultimate condition in this regard. The problem with assessing this feature adequately is that we do not always have sufficient samples of both sexes for some species of ingolfiellids. In males, the first pleopods always seem to be present.
36. Pleopod form
0 = biramous
1 = uniramous, narrow (Fig. 5I)
2 = uniramous, short fin
3 = uniramous, long fin (Fig. 5f)
Reductions of various sorts can be observed throughout the family Ingolfiellidae. Truly interstitial life of a certain mode might induce short appendages on the rear, or pleonal, end of a wriggling body that moves in a worm-like fashion. In this vein, the oceanic mud-dwelling species and the terrestrial cave-pool species would tend to have longer pleopods than the beach and riverine taxa that occupy open interstitial spaces between sand grains.
37. Uropod 1, rami
0 = free
1 = fused at peduncle
In Metaingolfiella, the aberrant species from a deep well in Italy, the rami are fused at the peduncle (1) In all other species in this analysis, the rami are free (0). This character provides no information to the phylogeny but is maintained to stress the peculiar situation in Metaingolfiella.
38. Uropod 1 and 2, size comparison
0 = uropods 1 and 2 not present
1 = subequal
2 = u2 larger than u1
3 = u1 larger than u2
This character measures the size of the first and second uropods relative to each other. No clear pattern emerges and although we consider these types of characters susceptible to allometric change, there is no alternative in dealing with these appendages. Age and molt stage might perhaps influence the relative size of the rami, but it is impossible to effectively take this into account. Our reasoning to include this feature here is that if there is a clear pattern, then we take the risk that it is due to coincidence; and if there is no pattern, then nothing is lost.
39. Uropod 1 and 2, total length
0 = no such uropods present
1 = long
2 = short
See comments vis-à-vis character 38.
40. Uropod 1, inner versus outer ramus length
0 = equal length
1 = outer ramus more than half of inner ramus length
2 = outer ramus less than half of inner ramus length
The variation in length of both rami of uropod 1 is not readily explained. No obvious function can be ascribed to this difference. It is possible that the outer ramus, which is in line with the underside of the body, should be longer to protect the inner ramus and the urosome from damage or to interact actively with other appendages (as was observed by Spooner, 1961, in grooming).
41. Uropod 3
0 = biramous
1 = uniramous
This character appears in Pseudoingolfiella, a member of the out-group but with a uniramous third uropod (1), and emphasises the most interesting role of such intermediate taxa.
42. Uropod 3 fusion
0 = right and left unfused
1 = fused peduncle
Only in Metaingolfiella are the peduncles fused to each other. The tendency towards fusing of elements in this species is three-fold now, given also the fusions already noted in the head and the rami of uropod 1. Tendency in this species seems to be towards fusion of adjacent elements into large units.
43. Telson form (Fig. 5l)
0 = well developed
1 = medium, fleshy
2 = short, flat
3 = short, fleshy
4 = short, bifurcate
A sturdy, well-developed telson is considered plesiomorphic with reference to the Amphipoda (Barnard & Karaman, 1983).
The following characters were also examined. They were not used in our final cladistic analysis, however, because there were too many question marks concerning them in the matrix. They all involve sexual differentiation, and because of the rarity of most ingolfiellideans it is not always possible to have adequate sample sizes that contain both sexes. We list them here, nevertheless, because at some point in the future, with better collections, it may become feasible to assess these features in a phylogenetic context across all species.
a. Gnathopod 2 male, reverse element on carpus
0 = absent
1 = present
At the end of the palmar margin, in the region of the large palmar setae delimiting the palm, an out-growth of irregular cuticular tissue often appears in older males (Fig. 6h)
b. Uropod 2 male, baso-facial spine
0 = absent
1 = present
In some species, a conspicuous, often somewhat hooked, spine is present on the basal part of the peduncle in the male. In other species, this is absent, no intermediate situation has been reported.
0 = present
1 = absent
All species in Ingolfiellidea would, by definition, be expected to have broodplates. But only few could be scored.