DOI: 10.5061/dryad.95x69p8zs
title: Data from: Developmental system drift in the patterning of the arthropod tarsus

description: The current understanding of proximodistal axis patterning in arthropod
 legs is grounded in insect models. The paradigm for appendage evolution in
 this phylum is that the gene regulatory network responsible for leg
 subdivision and patterning is broadly conserved. Recent surveys of these
 genes have suggested that chelicerate exemplars exhibit divergent
 appendage patterning dynamics, though functional data remain limited. One
 salient mismatch in expression occurs in homologs of the homeobox gene
 clawless. In insects, clawless is expressed in the distalmost leg
 territory, specifying the claw-bearing pretarsus. In the harvestman
 Phalangium opilio, clawless occupies a broad tarsal domain early in
 development, localizing later to the metatarsus-tarsus boundary,
 suggestive of a tarsal patterning function. Here, we tested the function
 of harvestman clawless using RNAi. Unlike insects, we show that clawless
 knockdown results in disrupted tarsal growth and patterning of its
 proximal segmental boundary, with no effect on the claw. Truncation of the
 tarsus is associated with defective tarsomere formation. We additionally
 surveyed the expression of clawless homologs in exemplars of chelicerate
 diversity, which suggests that the tarsal-patterning function for clawless
 was likely present in the most recent common ancestor of Chelicerata.
 These results join a suite of other comparative works suggesting that
 panarthropod appendage patterning exhibits numerous cases of developmental
 system drift.
description: # Data from: Developmental system drift in the patterning of the arthropod
 tarsus Dataset DOI:
 [10.5061/dryad.95x69p8zs](https://doi.org/10.5061/dryad.95x69p8zs) ##
 Description of the data and file structure The collected data consist of
 hyperstacks of confocal laser scanning microscopy images, in raw format.
 Each stack contains some or all of the following channels: 405 nm (blue);
 488 nm (green); 546 nm (red); 594 nm (red); 647 nm (far red). The
 numbering of the files corresponds the numbering of the figures in the
 associated paper. ### Files and variables #### File:
 FigureS6e.Plit_InsVI_03_le1WL_40x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S6e. Distal podomeres of leg 1 in the sea spider *Pycnogonum
 litorale* (instar VI) with expression of *clawless* and *aristaless*.
 Following complete separation of tarsus and propodus, only a small region
 of co-expression remains in the dorsal tarsus joint. The claw retains
 non-overlapping expression of *aristaless* in the dorsal compartment, and
 *clawless* in the ventral. In combination with expression surveys spanning
 postembryonic development, these data support conserved dynamics of both
 genes across Chelicerata, and the homology of arachnid metatarsus, tarsus,
 and pretarsus, with the sea spider tarsus, propodus, and claw. Channels
 correspond to DAPI (405nm), cuticular autofluorescence (488nm),
 *clawless* (594nm), and *aristaless* (647nm). #### File:
 Figure4c.S6d.Plit_InsV_16_ri1WL_40x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figures
 4c' and S6d. Leg 1 in the sea spider *Pycnogonum litorale* (instar V)
 with expression of *clawless* and *aristaless*. Following complete
 separation of tarsus and propodus, only a small region of co-expression
 remains in the dorsal tarsus joint. The claw retains non-overlapping
 expression of *aristaless* in the dorsal compartment, and *clawless* in
 the ventral. In combination with expression surveys spanning postembryonic
 development, these data support conserved dynamics of both genes across
 Chelicerata, and the homology of arachnid metatarsus, tarsus, and
 pretarsus, with the sea spider tarsus, propodus, and claw. Channels
 correspond to DAPI (405nm), cuticular autofluorescence (488nm),
 *clawless* (594nm), and *aristaless* (647nm). #### File:
 Figure4c.S6b.Plit_InsIV_65_HB_40x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figures
 4c and S6b. Ventral view of posterior body pole and leg 2 limb bud in the
 sea spider *Pycnogonum litorale* (instar IV) with expression of *clawless*
 and *aristaless*. Following outgrowth of the limb buds from the body wall,
 *aristaless* is expressed in a wide domain in the proximal limb bud and
 the distal terminus, whereas *clawless* occupies the intervening region
 with a sharp proximal border at the boundary of *aristaless*
 expression. In combination with expression surveys spanning postembryonic
 development, these data support conserved dynamics of both genes across
 Chelicerata, and the homology of arachnid metatarsus, tarsus, and
 pretarsus, with the sea spider tarsus, propodus, and claw. Channels
 correspond to DAPI (405nm), cuticular autofluorescence (488nm),
 *clawless* (594nm), and *aristaless* (647nm). #### File:
 FigureS6c.Plit_InsIV_54_ri1WL_40x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S6c. Leg 1 in the sea spider *Pycnogonum litorale* (instar IV) with
 expression of *clawless* and *aristaless*. At this stage, *clawless* is
 expressed in the tarsus-propodus precursor podomere and ventral
 compartment of the claw. *aristaless* is expressed diffusely in all
 further proximal podomeres, and strongly in the dorsal compartment of the
 claw. A narrow ring of co-expression occurs at the proximal boundary of
 *clawless* expression. In combination with expression surveys spanning
 postembryonic development, these data support conserved dynamics of both
 genes across Chelicerata, and the homology of arachnid metatarsus, tarsus,
 and pretarsus, with the sea spider tarsus, propodus, and claw. Channels
 correspond to DAPI (405nm), cuticular autofluorescence (488nm),
 *clawless* (594nm), and *aristaless* (647nm). #### File:
 FigureS5d.Plit_InsIV_54_OV_20x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S5d. Ventral view of the sea spider *Pycnogonum litorale* (instar IV) with
 expression of *clawless* and *aristaless*. Following outgrowth of the limb
 buds from the body wall, *aristaless* is expressed in a wide domain in the
 proximal limb bud and the distal terminus, whereas *clawless* occupies the
 intervening region with a sharp proximal border at the boundary of
 *aristaless* expression. In combination with expression surveys spanning
 postembryonic development, these data support conserved dynamics of both
 genes across Chelicerata, and the homology of arachnid metatarsus, tarsus,
 and pretarsus, with the sea spider tarsus, propodus, and claw. Channels
 correspond to DAPI (405nm), cuticular autofluorescence (488nm),
 *clawless* (594nm), and *aristaless* (647nm). #### File:
 FigureS6a.Plit_InsIII_82_lat_OV_40x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S6a. Lateral view of the sea spider *Pycnogonum litorale* (instar III)
 with expression of *clawless* and *aristaless*. Following outgrowth of the
 limb buds from the body wall, *aristaless* is expressed in a wide domain
 in the proximal limb bud and the distal terminus, whereas *clawless*
 occupies the intervening region with a sharp proximal border at the
 boundary of *aristaless* expression. In combination with expression
 surveys spanning postembryonic development, these data support conserved
 dynamics of both genes across Chelicerata, and the homology of arachnid
 metatarsus, tarsus, and pretarsus, with the sea spider tarsus, propodus,
 and claw. Channels correspond to DAPI (405nm), cuticular autofluorescence
 (488nm), *clawless* (594nm), and *aristaless* (647nm).  #### File:
 FigureS5b.Plit_InsIII_69_OV_40x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S5b. Ventral view of the sea spider *Pycnogonum litorale* (instar III,
 early) with expression of *clawless* and *aristaless*. Following outgrowth
 of the limb buds from the body wall, *aristaless* is expressed in a wide
 domain in the proximal limb bud and the distal terminus, whereas
 *clawless* occupies the intervening region with a sharp proximal border at
 the boundary of *aristaless* expression. In combination with expression
 surveys spanning postembryonic development, these data support conserved
 dynamics of both genes across Chelicerata, and the homology of arachnid
 metatarsus, tarsus, and pretarsus, with the sea spider tarsus, propodus,
 and claw. Channels correspond to DAPI (405nm), cuticular autofluorescence
 (488nm), *clawless* (594nm), and *aristaless* (647nm). #### File:
 FigureS5c.Plit_InsIII_68_OV_40x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S5c. Ventral view of the sea spider *Pycnogonum litorale* (instar III,
 late) with expression of *clawless* and *aristaless*. Following outgrowth
 of the limb buds from the body wall, *aristaless* is expressed in a wide
 domain in the proximal limb bud and the distal terminus, whereas
 *clawless* occupies the intervening region with a sharp proximal border at
 the boundary of *aristaless* expression. In combination with expression
 surveys spanning postembryonic development, these data support conserved
 dynamics of both genes across Chelicerata, and the homology of arachnid
 metatarsus, tarsus, and pretarsus, with the sea spider tarsus, propodus,
 and claw. Channels correspond to DAPI (405nm), cuticular autofluorescence
 (488nm), *clawless* (594nm), and *aristaless* (647nm). #### File:
 FigureS5a.Plit_InsII_34_OV_63x_Dapi.AF.al-647_Cll-594_TDE.lif
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S5a. Ventral view of the sea spider *Pycnogonum litorale* (instar II,
 late) with expression of *clawless* and *aristaless*. At this stage, both
 *clawless* and *aristaless* are detected in the leg 1 primordium flanking
 the posterior body pole. Both genes are partially co-expressed at the
 distal-most area of the primordium; the more restricted *Pl-al* territory
 is embedded in the wider cap-like *Pl-cll* expression domain. In
 combination with expression surveys spanning postembryonic development,
 these data support conserved dynamics of both genes across Chelicerata,
 and the homology of arachnid metatarsus, tarsus, and pretarsus, with the
 sea spider tarsus, propodus, and claw. Channels correspond to DAPI
 (405nm), cuticular autofluorescence (488nm), *clawless* (594nm), and
 *aristaless* (647nm).  #### File: Figure1b.WT.wholemount.al488.cll546.czi
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 1b. Embryo of the harvestman *Phalangium opilio* (stage 10) in ventral
 view with expression of *clawless* and *aristaless.* At this stage,
 *clawless* is expressed broadly throughout the distal appendage, whereas
 *aristaless* is detected in a ring domain proximal of the
 *clawless* territory, and weakly in the distal terminus. This broad
 *clawless* expression represents divergence from mandibulate expression
 patterns, providing initial evidence of developmental system drift in this
 lineage. Channels correspond to DAPI (405nm), *clawless* (546nm), and
 *aristaless* (647nm).  #### File: Figure1d.WT.wholemount.al488.cll546.czi
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 1d. Embryo of the harvestman *Phalangium opilio* (stage 13) in ventral
 view with expression of *clawless* and *aristaless*. At this stage,
 *clawless* expression localizes to a prominent ring domain at the
 metatarsus-tarsus podomere boundary. This expression pattern is suggestive
 of a role in podomere formation, rather than tarsal claw formation as in
 mandibulate arthropods. *aristaless* is detected in diffuse patches of
 expression in more proximal podomeres and the distal terminus. Channels
 correspond to DAPI (405nm), *clawless* (546nm), and *aristaless* (647nm). 
 #### File: Figure1c.WT.L2.al488.cll546.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure 1c. Leg 2 of the harvestman
 *Phalangium opilio* (stage 10) with expression of *clawless* and
 *aristaless*. At this stage, *clawless* is expressed broadly throughout
 the distal appendage, whereas *aristaless* is detected in a ring domain
 proximal of the *clawless* territory, and weakly in the distal terminus.
 This broad *clawless* expression represents divergence from mandibulate
 expression patterns, providing initial evidence of developmental system
 drift in this lineage. Channels correspond to DAPI (405nm), *clawless*
 (546nm), and *aristaless* (647nm). #### File:
 Figure1e.WT.L2.al488.cll546.czi **Description:** Confocal microscopy
 hyperstack corresponding to Figure 1e (*Phalangium opilio*). Leg II of the
 harvestman *Phalangium opilio* (stage 13) with expression of *clawless*
 and *aristaless*. At this stage, *clawless* expression localizes to a
 prominent ring domain at the metatarsus-tarsus podomere boundary. This
 expression pattern is suggestive of a role in podomere formation, rather
 than tarsal claw formation as in mandibulate arthropods. *aristaless* is
 detected in diffuse patches of expression in more proximal podomeres and
 the distal terminus. Channels correspond to DAPI (405nm), *clawless*
 (546nm), and *aristaless* (647nm).  #### File:
 Figure2a.WT.wholemount.Dl546.al647.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure 2a. Negative control embryo
 of *Phalangium opilio* (stage 11) with expression of
 *Delta* and *aristaless*. Note that at this stage, *Delta* is expressed in
 multiple ring domains in the tarsus, indicative of sequential tarsomere
 formation. Channels correspond to DAPI (405nm), *Delta* (546nm), and
 *aristaless* (647nm).  #### File:
 Figure2b.cllRNAi.wholemount.dl546.al647.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure 2b. Embryo of *Phalangium
 opilio* (stage 11) following RNAi against *clawless* with expression
 of *Delta* and *aristaless*. In contrast to negative control embryos,
 depletion of *clawless* yields disruptions to *Delta* ring domains in the
 tarsus, consistent with abolished tarsomere formation. Lingering patches
 of *Delta* expression correspond to putative sensory structures. Channels
 correspond to DAPI (405nm), *Delta* (546nm), and *aristaless* (647nm).
 #### File: Figure2c.WT.Leg2.dl546.al647.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure 2c. Leg 2 of negative
 control *Phalangium opilio* embryo (stage 11) with expression of *Delta*
 and *aristaless*. Note that at this stage, *Delta* is expressed in
 multiple ring domains in the tarsus, indicative of sequential tarsomere
 formation, with the highest intensity expression in the proximal tarsus.
 Channels correspond to DAPI (405nm), *Delta* (546nm), and *aristaless*
 (647nm).  #### File: Figure4a.S4c.Alon.al.cll.czi
 **Description:** Confocal microscopy hyperstack corresponding to Figures
 4a and S4c. Embryo of the acariform mite *Archegozetes longisetosus* in
 lateral view with expression of *clawless* and *aristaless*. At this
 stage, *clawless* exhibits a broad cap-like expression domain beyond the
 *aristaless*-positive cells in the distal terminus. *aristaless* is
 likewise expressed in a small patch in the proximal territory of each
 appendage. This *clawless* domain is comparable to the expression domain
 of *clawless* in early* P. opilio* appendage development, suggestive of
 conserved dynamics across Chelicerata. Channels correspond to DAPI
 (405nm), *clawless* (546nm), and *aristaless* (647nm).  #### File:
 Figure2e.WT.L2.odd488.Dl546.czi **Description:** Confocal microscopy
 hyperstack corresponding to Figure 2e. Leg II of a negative control
 *Phalangium opilio* embryo with expression of *Delta* and *odd-skipped*.
 Note the prominent rings of *odd-skipped* expression at developing
 podomere boundaries and ring domains of *Delta* indicative of sequential
 tarsomere addition. Channels correspond to DAPI (405nm), *odd-skipped*
 (488nm), and *Delta* (546nm).  #### File:
 Figure4d.Parhyale.wholemount.cll1546_SoxN647.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure 4d. Embryo of the amphipod
 *Parhyale hawaiensis* (stage 20) with expression of *clawless-1*. At this
 stage, *clawless-1* is expressed in the distal territory of both antennal
 pairs and thoracopods 1-8, and in small domains in the central nervous
 system. Note that expression in the thoracopods exhibits two discrete
 patches. Channels correspond to DAPI (405nm) and *clawless-1* (546nm).  
 #### File: Figure4e.Parhyale.wholemount.cll2546_soxN647_S20.czi
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 4e. Embryo of the amphipod *Parhyale hawaiensis* (stage 22) with
 expression of *clawless-2*. Both *clawless* paralogs in this species
 exhibit comparable dynamics, with *clawless-2* expressed distally in both
 antennal pairs and thoracopods 1-8, and the central nervous system, albeit
 more homogeneously than *clawless-1.* Channels correspond to DAPI (405nm)
 and *clawless-2* (546nm).  #### File:
 Figure2d.cllRNAi.Leg2.dl546.al647.czi **Description:** Confocal microscopy
 hyperstack corresponding to Figure 2d. Leg 2 of *clawless* RNAi
 *Phalangium opilio* embryo (stage 11) with expresion of *Delta* and
 *aristaless*. Note the prominent swelling of the appendage at the putative
 metatarsus-tarsus boundary, consistent with the inferred role in podomere
 formation. Disruptions of *Delta* rings in the tarsus are also consistent
 with failed tarsomere formation. Channels correspond to DAPI (405nm),
 *Delta* (546nm), and *aristaless* (647nm).   #### File:
 Figure4f.Parhyale.al1488_cll1546_SoxN647.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure 4f. Detail of thoracopods in
 an embryo of the amphipod *Parhyale hawaiensis* (stage 22) with expression
 of *clawless-1*.  At this stage, *clawless-1* is expressed in the distal
 territory of both antennal pairs and thoracopods 1-8, and in small domains
 in the central nervous system. Note that expression in the thoracopods
 exhibits two discrete patches in the dactylus and propodus. Channels
 correspond to DAPI (405nm) and *clawless-1* (546nm). #### File:
 Figure2f.cllRNAi.Leg2.odd488.Dl546.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure 2f. Leg 2 of a *clawless*
 RNAi *Phalangium opilio* embryo with expression of *Delta* and
 *odd-skipped.* Note the absence of the distalmost ring of *odd-skipped* in
 comparison to negative control embryo, consistent with a fusion of
 metatarsus and tarsus. Disruptions to tarsal rings of *Delta* again
 coincide with abolished tarsomere addition. Channels correspond to DAPI
 (405nm), *odd-skipped* (488nm), and *Delta* (546nm).  #### File:
 FigureS4d_Alon_al_cll.czi **Description:** Confocal microscopy hyperstack
 corresponding to Figure S4d. Embryo of the acariform mite *Archegozetes
 longisetosus* in lateral view with expression of *clawless* and
 *aristaless.* In later stages of development, *clawless* retains strong
 expression in a broad cap-like domain in the distal appendage, whereas
 *aristaless* is retained in the distal terminus but is weaker and more
 diffuse in the proximal appendage. This *clawless* domain is comparable to
 the expression domain of *clawless* in early* P. opilio* appendage
 development, suggestive of conserved dynamics across Chelicerata. Channels
 correspond to DAPI (405nm), *clawless* (546nm), and *aristaless* (647nm). 
 #### File: Figure4b.Isca.st13_cll_al.lif **Description:** Confocal
 microscopy hyperstack corresponding to Figure 4b. Embryos of the
 parasitiform tick *Ixodes scapularis* with expression of *clawless.* Note
 prominent localization of *clawless* expression to the boundary of basi-
 and telotarsus podomeres, similar to the late localization of *clawless*
 to metatarsus-tarsus boundary in *P. opilio.* This expression is again
 consistent with conserved *clawless* dynamics across Chelicerata. Channels
 correspond to DAPI (405nm) and *clawless* (546nm).  #### File:
 FigureS4a_Alon_al_cll.czi **Description:** Confocal microscopy hyperstack
 corresponding to Figure S4a. Embryo of the acariform mite *Archegozetes
 longisetosus* in lateral view with expression of *clawless* and
 *aristaless*. At this stage, *clawless* is broadly expressed in the
 appendage primordia. In contrast, *aristaless* expression was not
 detected. Channels correspond to DAPI (405nm), *clawless* (546nm), and
 *aristaless* (647nm).  #### File:
 FigureS1a.WT.wholemount.odd488.cll546.dll647.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure S1a. Negative control
 *Phalangium opilio* embryo (stage 10) with expression of *clawless,
 odd-skipped,* and *Distal-less*. Note prominent *clawless* expression in
 the distal appendage. Channels correspond to DAPI *(405nm), odd-skipped*
 (488nm), *clawless* (546nm), and *Distal-less* (647nm).  #### File:
 FigureS1b.cllRNAi.wholemount.odd488.cll546.dll647.czi
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S1b. *clawless* RNAi *Phalangium opilio* embryo with expression of
 *clawless, odd-skipped,* and *Distal-less*. RNAi experiments yield
 on-target depletion of *clawless*, whereas expression of *odd-skipped* and
 *Distal-less* remains. Channels correspond to DAPI (405nm), *odd-skipped*
 (488nm), *clawless* (546nm), and *Distal-less* (647nm).  #### File:
 FigureS4b_Alon_al_cll.czi **Description:** Confocal microscopy hyperstack
 corresponding to Figure S4b. Embryo of the acariform mite *Archegozetes
 longisetosus* in lateral view with expression of *clawless* and
 *aristaless*. At this stage, *clawless* exhibits a broad cap-like
 expression domain beyond the *aristaless*-positive cells in the distal
 terminus. *aristaless* is likewise expressed in a small patch in the
 proximal territory of each appendage. This *clawless* domain is comparable
 to the expression domain of *clawless* in early* P. opilio* appendage
 development, suggestive of conserved dynamics across Chelicerata. Channels
 correspond to DAPI (405nm), *clawless* (546nm), and *aristaless* (647nm). 
 #### File: Figure4g.Parhyale_al2488_cll2546_SoxN647.czi
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 4g. Detail of thoracopods in an embryo of the amphipod *Parhyale
 hawaiensis* (stage 24) with expression of *clawless-2*. At this stage,
 *clawless-2* is expressed comparably to *clawless-1*, in the distal
 territory of both antennal pairs and thoracopods 1-8, and in small domains
 in the central nervous system. Unlike slightly earlier stages,
 *clawless-2* is expressed in two discrete patches. Channels correspond to
 DAPI (405nm) and *clawless-2 *(546nm). #### File:
 FigureS4e_Alon_al_cll.czi **Description:** Confocal microscopy hyperstack
 corresponding to Figure S4e. Embryo of the acariform mite *Archegozetes
 longisetosus* in lateral view with expression of *clawless* and
 *aristaless.* In later stages of development, *clawless* retains strong
 expression in a broad cap-like domain in the distal appendage, whereas
 *aristaless* is retained in the distal terminus but is weaker and more
 diffuse in the proximal appendage. This *clawless* domain is comparable to
 the expression domain of *clawless* in early* P. opilio* appendage
 development, suggestive of conserved dynamics across Chelicerata. Channels
 correspond to DAPI (405nm), *clawless* (546nm), and *aristaless* (647nm). 
 #### File: FigureS3a_WT_St9_ser488_Dl546.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure S3a. Embryo of *Phalangium
 opilio* (stage 9) in ventral view with expression of *Serrate* and
 *Delta.* To determine whether progressive rings of *Delta* expression
 coincide with tarsomere addition, representative stages of leg elongation
 were surveyed. At this early stage, *Serrate* is expressed in ring domains
 corresponding to putative podomere boundaries, whereas *Delta* is
 expressed prominently in the central nervous system, and in a weak ring
 domain in the leg 2 tarsus. As leg 2 will bear the highest number of
 tarsomeres in this lineage, this early expression may be indicative of
 earlier onset of tarsomere addition. Channels correspond to DAPI (405nm),
 *Serrate* (488nm), and *Delta* (546nm).  #### File:
 FigureS2.cllRNAi.wholemount.dl546.al647.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure S2. *clawless* RNAi
 *Phalangium opilio* embryo (stage 11) with expression of *clawless* and
 *aristaless*. A subset of RNAi embryos exhibited a pronounced bend or kink
 in the distal appendage, attributed to asymmetric projections of tissue at
 the malformed joint between metatarsus and tarsus. Channels correspond to
 DAPI (405nm), *clawless* (546nm), and *aristaless* (647nm).  #### File:
 FigureS3c_WT_St11_Ser488_Dl546.czi **Description:** Confocal microscopy
 hyperstack corresponding to Figure S3c. Embryo of *Phalangium
 opilio* (stage 11) in ventral view with expression of *Serrate* and
 *Delta.* To determine whether progressive rings of *Delta* expression
 coincide with tarsomere addition, representative stages of leg elongation
 were surveyed. At this stage, *Serrate* is still expressed in ring domains
 corresponding to putative podomere boundaries, whereas *Delta* is
 expressed prominently in the central nervous system, and in several ring
 domains in all leg tarsi. The increased number of rings relative to stage
 10 embryos, suggests *Delta* demarcates the sequentially added tarsomeres.
 Note the absence of *Delta* rings in the pedipalpal tarsi, consistent with
 their lack of tarsomeres. Channels correspond to DAPI (405nm), *Serrate*
 (488nm), and *Delta* (546nm).  #### File:
 FigureS3b_WT_wholemount_St10_Ser488_Dl546.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure S3b. Embryo of *Phalangium
 opilio* (stage 1o) in ventral view with expression of *Serrate* and
 *Delta.* To determine whether progressive rings of *Delta* expression
 coincide with tarsomere addition, representative stages of leg elongation
 were surveyed. At this stage, *Serrate* is still expressed in ring domains
 corresponding to putative podomere boundaries, whereas *Delta* is
 expressed prominently in the central nervous system. In the tarsi of legs
 1, 3, and 4, *Delta* exhibits one strong ring domain, while exhibiting
 several such domains in the leg 2 tarsi. The increased number of rings in
 leg 2 relative to stage 9 embryos, suggests *Delta* demarcates the
 sequentially added tarsomeres. Note the absence of *Delta* rings in the
 pedipalpal tarsi, consistent with their lack of tarsomeres. Channels
 correspond to DAPI (405nm), *Serrate* (488nm), and *Delta* (546nm).  ####
 File: FigureS3d_WT_St12_Ser488_Dl546.czi **Description:** Confocal
 microscopy hyperstack corresponding to Figure S3d. Embryo of *Phalangium
 opilio* (stage 12) in ventral view with expression of *Serrate* and
 *Delta.* To determine whether progressive rings of *Delta* expression
 coincide with tarsomere addition, representative stages of leg elongation
 were surveyed. At this stage, *Serrate* is still expressed in ring domains
 corresponding to putative podomere boundaries, whereas *Delta* is
 expressed prominently in the central nervous system, and in numerous ring
 domains in all leg tarsi. The increased number of rings relative to stage
 11 embryos, suggests *Delta* demarcates the sequentially added tarsomeres.
 Note the absence of *Delta* rings in the pedipalpal tarsi, consistent with
 their lack of tarsomeres. Channels correspond to DAPI (405nm), *Serrate*
 (488nm), and *Delta* (546nm).  #### File:
 FigureS3e_WT_L2_stage10_Dl546.czi **Description:** Confocal microscopy
 hyperstack corresponding to Figure S3e. Detail of distal leg 2
 in *Phalangium opilio* embryo (stage 10) with expression of *Delta.* Note
 high intensity of expression in rings distal to the tarsus boundary, the
 putative location of tarsomere addition. Channels correspond to DAPI
 (405nm) and *Delta* (546nm).  #### File: FigureS3f_WT_L2_stage11_Dl546.czi
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S3f (*Phalangium opilio*). Detail of distal leg 2 in *Phalangium opilio*
 embryo (stage 11) with expression of *Delta.* The increase in number of
 *Delta* rings compared to stage 10 is indicative of demarcating
 sequentially added tarsomeres.**** Channels correspond to DAPI (405nm) and
 *Delta* (546nm).  #### File: FigureS3g_WT_L2_stage12_Dl546.czi
 **Description:** Confocal microscopy hyperstack corresponding to Figure
 S3g. Detail of distal leg 2 in *Phalangium opilio* embryo (stage 12) with
 expression of *Delta.* Note that in later stages, *Delta* rings are
 obscured by expression localized to putative sensory cells. Channels
 correspond to DAPI (405nm) and *Delta* (546nm).  ## Code/software All .czi
 and .lif files are readily viewed in Fiji or a similar image processing
 software (e.g., Zeiss Zen Lite).