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\begin{document}

\title{Land cover change affects the amount and reactivity of DOM exported from
old growth and regenerating forests in headwater ecosystems}



\author[1]{Timothy Fegel}%
\author[2]{Claudia Boot}%
\author[2]{Tim Covino}%
\author[1]{Kelly Elder}%
\author[2,2]{Ed Hall}%
\author[1]{Banning Starr}%
\author[1]{Charles Rhoades}%
\affil[1]{USDA Forest Service Rocky Mountain Research Station}%
\affil[2]{Colorado State University}%


\vspace{-1em}



  
  \date{June 02, 2024}


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\selectlanguage{english}
\begin{abstract}
Headwater forest ecosystems of the western U.S. generate a large portion
of the dissolved organic matter (DOM) transported across North America.
Land cover changes that alter forest structure and forest species
composition affect the quantity and composition of DOM transferred to
aquatic ecosystems. Clear-cut harvesting effects \textasciitilde{}1\% of
the forest area of North America annually, leaving most forests in
varying stages of successional regrowth, and the total area of
old-growth forest decreasing. The consequences of this widespread
management practice on watershed carbon cycling remain unknown. We
investigated the role of land cover change from old-growth subalpine
forest to lodgepole pine dominated second-growth on the character and
reactivity of DOM hillslope exports. We evaluated inputs of DOM from
litter leachates and export of DOM collected at the base of trenched
hillslopes during a three-year period (2016-2018) at the Fraser
Experimental Forest in northcentral Colorado, USA. Dissolved organic
carbon (DOC) and total dissolved nitrogen (TDN) were higher in lateral
subsurface flow draining old- versus secondary-growth forest.
Fluorescence spectroscopy showed that the DOM exported from the
old-growth forest was more heterogeneous and aromatic and that
proteinaceous, microbially processed DOM components were more prevalent
in the second-growth forest. Biological oxygen demand (BOD) assays
revealed much lower microbial metabolism of both DOM inputs from litter
leachate and subsurface exports from old-growth forest. Old-growth and
second-growth forests are co-mingled in managed ecosystems, and our
findings demonstrate that the influence of species composition on DOM
inputs can affect the reactivity of DOM transferred from terrestrial to
aquatic ecosystems.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Dissolved organic matter (DOM) derived from vegetation and soils
accounts for up to 90\% of the carbon (C) found in surface water
(Aitkenhead-Peterson, McDowell, \& Neff, 2003) and DOM character
regulates microbial C processing in aquatic environments (Benner, 2003;
D'Andrilli, Junker, Smith, Scholl, \& Foreman, 2019; Lennon \& Pfaff,
2005). While encompassing less than 1\% of the global land surface,
inland waters regulate carbon cycling from landscape to continental
scales (Battin et al., 2009; Cavallaro et al., 2018; Cole et al., 2007;
Raymond et al., 2013), and headwater streams drain 70\% of North
American land area (Colvin et al., 2019). Changes to forest land cover
that affect terrestrial DOM character will influence aquatic ecosystem
metabolism (Cawley, Yamashita, Maie, \& Jaffe, 2014; Lajtha \& Jones,
2018; Williams, Yamashita, Wilson, Jaffe, \& Xenopoulos, 2010), and
deciphering these relationships are crucial for predicting C cycling in
inland waters.

Vegetation responses to land management can alter watershed hydrology
and biogeochemistry for decades to centuries (Chantingy, 2003; Lee \&
Lajtha, 2016; Rhoades, Hubbard, \& Elder, 2017; Troendle \& King, 1985;
Wilm \& Dunford, 1948). In the mountain ranges of the American West,
where the short growing season and cold, dry climate limit forest
regrowth, the consequences of timber harvesting on snowpack, streamflow,
and nutrient export as well as on potential wildfire behavior and
susceptibility to insect outbreaks are long-lasting (Stottlemyer \&
Troendle, 1999; Rhoades et al., 2017; Wilm \& Dunford, 1948). Timber
harvesting decreases canopy interception and evapotranspiration
resulting in more snowpack water storage, and increased soil moisture
and streamflow (Troendle \& King, 1985) with effects that remain
measurable for more than 50 years. Timber harvesting also shifts the
species composition of these forests and their development trajectory
for a more than a century (Collins, Rhoades, Hubbard, \& Battaglia,
2011; Lotan \& Perry, 1983).

Harvest of subalpine forests comprised of subalpine fir (\emph{Abies
lasiocarpa} ), Engelmann spruce (\emph{Picea engelmannii} ) and
lodgepole pine (\emph{Pinus contorta} )~typically regenerate into
lodgepole pine-dominated stands (Collins et al., 2011). While the
effects of land cover, age, and species change on terrestrial nutrient
cycling and organic matter decomposition are well studied (Chantigny,
2003 for a review), their consequences on adjacent aquatic ecosystem C
cycling are not. The chemical characteristics of litter in regenerating
forests alter hillslope DOM inputs relative to those in old-growth
forest (Beggs \& Summers, 2011; D'Andrilli et al., 2019), likely
regulating the reactivity of DOM exported to streams at watershed
scales. The highly polyphenolic and protein-like DOM released from
lodgepole pine litter in regenerating stands, for example, is more
reactive than that generated by old-growth, mixed-conifer forests (Beggs
\& Summers, 2011; Yavitt \& Fahey, 1984 \& 1986). Connecting the effects
of land cover type on the reactivity of DOM inputs from the forest floor
to the reactivity of hillslope DOM exports will increase understanding
of the effects of land cover change on watershed-scale C cycling.

The consequences of land cover and tree species shifts on
watershed-scale DOM dynamics benefit from techniques that differentiate
DOM reactivity of hillslope inputs and exports. Analysis of fluorescing
components of DOM (FDOM) has become a relatively routine, low-cost
approach to characterize the relative abundance of biologically reactive
organic molecules (Smith et al. 2018). Correlation between fluorescing
and non-fluorescing DOM molecules of similar chemical structures and
biological reactivities expands the utility of FDOM as an index of
overall DOM quality (Stubbins et al., 2014).~Optical characterization
can be used in conjunction with heterotrophic microbial oxygen
consumption assays to identify which DOM components are microbially
created or utilized (D'Andrilli et al., 2019).

In this study we compare DOM inputs from litter leachate and the
concentration, character and biological reactivity of DOM exports in
subsurface flow between adjacent old-growth subalpine and second-growth
pine forests. Early work at these sites documented that clear cut
harvesting resulted in lasting increases in snow accumulation,
subsurface discharge, and nutrient export (Reuss, Stottlemyer, \&
Troendle, 1997; Starr, 2004; Troendle \& Reuss, 1997). The amount of DOC
exported from forests is determined by O horizon C content, with
old-growth forests exporting more DOC compared to regenerating ones
(Cawley et al., 2014; Lajtha \& Jones, 2018; Pacific, Jensco, \&
McGlynn, 2010). However, DOC reactivity is affected by C character and
processing within subsurface flow paths (Lehmann \& Kleber, 2015).
Because old-growth forests generally have deeper O-horizons composed of
more recalcitrant DOM (Jandle et al., 2007; Johnson, Johnson,
Huntington, \& Siccama, 1991), we expect greater flux but lower
biological reactivity of DOC from these forests. Conversely, we
hypothesize that DOM exported from pine-dominated, second-growth forest
to contain proportionally more biologically reactive DOM typical of the
water-soluble proteins and polyphenolics found in pine needle litter
(Beggs \& Summer, 2011). Western conifer forests often require a century
to regrow to their pre-harvest stand structure (Burns \& Honkala, 1990),
so the extent of old-growth forest cover has decreased during more than
a century of timber harvesting and most forests are in intermediate
stages of recovery (Anderson-Teixeira et al., 2013; Hurtt et al., 2011;
McDowell et al., 2020). This study evaluates how forest change following
clear-cut harvesting alters the reactivity of DOM from the landscape to
aquatic ecosystems.

\textbf{Methods}

Site description

This research occurred at the Fraser Experimental Forest (FEF), 137 km
west of Denver, Colorado. Mean annual temperature at FEF headquarters
(elevation 2725 m) is 0.5 \selectlanguage{ngerman}°C, and ranges from -10 °C in January to 12.7
°C in July. Mean annual precipitation is 610 mm (range 427-902 mm;
1981-2010), nearly two-thirds of which falls as snow from October to
May. Annual inorganic N deposition averaged 2.6 kg N
ha\textsuperscript{-1} over the last decade (Argerich et al., 2013). The
Fraser area was extensively glaciated and FEF is underlain by
metamorphosed rock, most commonly biotite schist and hornblende or
calc-silicate gneiss (Shroba, Bryant, Kellogg, Theobald, \& Brandt,
2010). Soils are skeletal, sandy loam Dystric and Typic Cryochrepts
(Alstatt \& Miles, 1983) with 20-30\% gravel and 30-50\% cobble-sized
materials. To evaluate the role of forest cover on hillslope hydrologic
processes, including quantifying the timing and amount of subsurface
flow, paired trenched hillslopes were installed in 1979. The paired
hillslopes are adjacent to each other on a glacial moraine that is
underlain at 3 m by a clay aquaclude (Reuss, Stottlemyer, \& Troendle,
1997; Troendle \& Reuss, 1997). Subsurface flow is collected by slotted
PVC pipe installed at 4 m depth in lined trenches at the base of the
hillslopes and piped to individual gauging stations (HS flumes) where
water level is recorded using Druck pressure transducers and Campbell
CR-1000 data loggers at 10-minute intervals. These water level data are
then converted to subsurface flow rates using known relations between
water depth and discharge (Bos, 1989). In December 1984, after five
years of pretreatment calibration of subsurface flow rates, a 3-ha
section of one of the trenched hillslopes was clear cut harvested, while
the other was left intact.

Old-growth subalpine forests at FEF are comprised of equal proportions
of lodgepole pine (\emph{Pinus contorta} ), subalpine fir (\emph{Abies
lasiocarpa} ) and Englemann spruce (\emph{Picea engelmannii} ),
respectively 32\%, 34\%, and 34\% of overstory basal area (Supp. Info:
T1). Low-stature shrubs (\emph{Vaccinium scoparium, V. myrtillus, V.
caespitosum, Shepherdia canadensis} ) form a dense understory beneath
the coniferous overstory (Popovich, 1993). The clear-cut hillslope has
regenerated into a pine-dominated second-growth stand (82\% of overstory
basal area) with 9 cm mean diameter and 4 m mean height (Supp. Info:
T1). Mountain pine bark beetles (\emph{Dendroctonus ponderosae} )
preferentially attack larger trees (Rhoades et al., 2017), and in a
recent outbreak beetles killed 18\% of the old-growth basal area but did
not infest the second-growth stand.

Sampling and analysis

Lateral subsurface flow begins mid to late April at the start of
snowmelt, increases to the peak in early to mid-May, then declines and
halts by mid to late June (Fig 1a, Troendle \& Reuss, 1997). We
collected hillslope export samples as lateral subsurface flow at the
base of each hillslope (i.e., at the piped trenches) weekly in 2016-2018
and analyzed for nutrients, dissolved organic carbon (DOC), total
dissolved nitrogen (TDN), and DOM chemical characteristics. These
samples were collected in pre-combusted (heated for 3 hours at 500 \selectlanguage{english}0C)
amber glass bottles then filtered through 0.7 \selectlanguage{greek}µ\selectlanguage{english}m pore-size glass fiber
filters (Millipore Corp, Burlington, MA) within 24 hours of collection
and analyzed for DOC, TDN, and DOM fluorescence spectroscopy. We
collected DOM reactivity samples in 250 mL pre-combusted amber glass
bottles, then filtered through (GF/C Whatman\selectlanguage{ngerman}®, 1.2 \selectlanguage{greek}µ\selectlanguage{english}m effective pore
size) filters within 3 hours of collection to remove bacterial grazers.
DOC and TDN were determined using a Shimadzu
TOC-V\textsubscript{CPN}total organic carbon analyzer, with 2M HCl
addition before analysis to remove mineral C (Shimadzu Corporation
Columbia, MD). Detection limits for DOC and TDN were 50 \selectlanguage{greek}µ\selectlanguage{english}g
L\textsuperscript{-1}.

The chemical character of DOM exported from each hillslope (i.e.,
lateral subsurface flow) was analyzed using UV-Visible fluorescence
spectroscopy on a Horiba Scientific Aqualog (Horiba-Jobin Yvone
Scientific Edison, New Jersey, US). Sample C concentration was
standardized to 5 mg C L\textsuperscript{-1} using deionized water
(\textgreater{}18 m). Ultraviolet absorbance was analyzed at the 254 nm
wavelength. Excitation emission matrices (EEMs) fluorescence scans were
completed from 240 nm-600 nm excitation and emission wavelengths, with 3
nm band-pass, 3 nm increments, and 3 second integrations. Scans were
blank corrected using deionized water and corrected for inner filter
effects (Kubista, Sjoback, Eriksson, \& Albinsson, 1994). First- and
second-order Rayleigh scattering were masked (10 nm width masking), and
samples were normalized by the area of the deionized water Raman
scattering peak (Lawaetz \& Stedmon, 2009).

SUVA\textsubscript{254} (L mg
C\textsuperscript{-1}m\textsuperscript{-1}) was calculated by dividing
ultraviolet absorbance at 254 nm by DOC concentration (Weishaar et al.,
2003). Humification index (HIX) was calculated as the area of spectra
collected at 254 nm emission under 435-480 nm excitation divided by the
area under the peak 300-345 nm emission (Zsolnay, Baigar, Jimenez,
Steinweg, \& Saccomandi, 1999). Fluorescence Index (FI) was calculated
as the ratio between emission at 470 nm and 520 nm at 370 nm excitation
(McKnight et al., 2001; Cory \& McKnight, 2005). Fluorescence regional
integration modelling (FRI) was applied to EEMs data to account for
areas of increased fluorescence not identified by standard EEMs indices
(Chen, Westerhoff, Leenheer, \& Booksh, 2003). Briefly, EEMs scans were
separated into five regions associated with distinct DOM chemical
components. Total fluorescence intensity for each region was divided by
total intensity of the entire scan, resulting in percentages of total
FDOM derived from each DOM chemical component region.

We sampled the organic (O horizon) and mineral (A horizon; 10 cm depth)
soil layers at ten locations distributed across the old-growth and
second-growth hillslopes (n=20). O horizon material was sampled within a
30 cm by 30 cm quadrat and total O-horizon depth was measured in each
quadrat corner. O horizons were separated into litter and duff layers as
follows (FIA 2019): needles and recognizable plant material (\textless{}
6 mm diameter) were classified as litter, while unrecognizable,
fragmented material between the litter and mineral soil layers was
considered duff. There was no measurable duff in the second-growth
forest. Mineral soil (0-10 cm depth) was collected using a 7.5 cm
diameter corer after the O horizon was removed. Mineral soils had rocks,
mosses, and lichens removed and were then sieved to 2 mm. We determined
gravimetric soil moisture after drying a subsample at 105 0\selectlanguage{english}C for 48
hours. Organic horizon samples were well mixed and moss, lichen, and
rocks were removed by hand. Organic horizon samples were dried for 6
days at 60 0\selectlanguage{english}C and the total mass was recorded. Total bulk density for
mineral and organic soils was calculated by dividing the dry weight of
the total soil (including \textgreater{} 2 mm fraction in mineral soils)
by the sample volume. A subset of each sample was dried for 48 hours at
60 0\selectlanguage{english}C, then ground and analyzed for total C and N by dry combustion
(LECO 1000 CHN analyzer, LECO Corporation, St. Joseph, MI, USA). Total C
and N pools were calculated by multiplying C and N concentrations by the
associated O- and A-horizon masses. Water extractable soil organic
matter (WEOM) samples were created by leaching a litter layer sample
from each hillslope's O-horizon to replicate DOM inputs (Sparling,
Vojvodic-Vukovic, \& Schipper, 1998). Litter samples were air dried for
3 days then 10 g subsamples were steeped in 50 mL of DI
(\textgreater{}18 m) at 70 0\selectlanguage{english}C for 18 hours, shaken and filtered through
0.7 \selectlanguage{greek}µ\selectlanguage{english}m pore size glass fiber filters.

Biological oxygen demand assays were performed to assess the reactivity
of DOM inputs (WEOM litter leachates) and DOM exports (lateral
subsurface flow). DOM input incubations were diluted and standardized to
30 mg C L\textsuperscript{-1} to mirror C concentrations of DOM export
incubations. Each DOM input sample was inoculated with 88 mL of water
from an adjacent stream (Sparling et al. 1998), and export DOM was
incubated with microbes that occurred naturally in the subsurface flow.
Relationships in reactivity between input and output DOM should be
considered tentative correlations, as different microbial cultures were
used in litter and subsurface leachate incubations. Experimental
controls, which only contained the stream water microbial culture with
no additional DOM and analytical controls which only contained DI
(\textgreater{}18 m) were incubated simultaneously with the oxygen
demand assays. Microbial dissolved oxygen consumption (mg
O\textsubscript{2} L\textsuperscript{-1}) was continuously measured (15
second intervals) using Oxy-4 probes (PreSens, Precision Sensing GmbH,
Regensburg, Germany). Input and export samples were incubated in the
dark at 20 0\selectlanguage{english}C and the hourly oxygen consumption rate (mg
O\textsubscript{2} L\textsuperscript{-1} hr\textsuperscript{-1}) was
averaged for each sample over the 24-hour incubation period. No samples
approached hypoxic conditions (\textless{}4 mg
O\textsubscript{2}L\textsuperscript{-1}). Oxygen consumption rates were
normalized per gram of C to remove any bias of C quantity on consumption
rate. Post-incubation samples were filtered to remove microbial biomass
(Nucleopore\selectlanguage{ngerman}®, polycarbonate filter, 0.2 \selectlanguage{greek}µ\selectlanguage{english}m). Concentration of C
mineralized (\selectlanguage{greek}Δ \selectlanguage{english}mg C L\textsuperscript{-1}) was calculated as the
difference between measured pre- and post- incubation DOC
concentrations, then converted to loss as
CO\textsubscript{2}(\selectlanguage{greek}Δ\selectlanguage{english}CO\textsubscript{2}, in mg CO\textsubscript{2}
L\textsuperscript{-1}), under the assumption all CO\textsubscript{2}
produced by incubations was removed from the sample with HCl addition
during C measurement. The respiratory quotient (RQ), was calculated by
dividing C lost to respiration (\selectlanguage{greek}Δ\selectlanguage{english}CO\textsubscript{2}, in mg
CO\textsubscript{2}L\textsuperscript{-1}) by the change in O during the
incubation (\selectlanguage{greek}Δ\selectlanguage{english}O\textsubscript{2} in mg O\textsubscript{2}
L\textsuperscript{-1}).

DOC and TDN concentrations, and FDOM indices in subsurface flow exports
were compared between old-growth and second-growth land cover types
using Welch-Satterthwaite unequal variance t-test with significance
assigned at p \textless{} 0.01 (R Core Team, 2019). Regional percentages
for FDOM components (FRI modelling) in export samples were logit
transformed before statistical analyses to remove biases inherent in
proportional data. We compared input nutrient concentrations and pools
in organic and mineral horizon soils, along with comparisons of oxygen
consumption and respiratory quotients in DOM input and export samples
using the Student's t-tests for parametric data, with significance
assigned at p \textless{} 0.01 (t.test function, R Core Team, 2019). The
data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{Results}

C and N inputs to the hillslope from soil O horizons

The old-growth hillslope contained 6, 4, and 11 times more O horizon
mass, total C, and total N content compared to the adjacent
second-growth stand, respectively (Table 1). The C:N ratio of the
old-growth total O horizon was roughly half that of the second-growth
hillslope. Mass and C content of litter layers were similar between
hillslopes but the N concentration was twice as high in litter on the
old-growth hillslope. The C concentration of the uppermost mineral soil
(10cm depth) was 1.3 times higher in the second-growth forest but N
concentration did not differ (Table 1).

C and N exports in lateral subsurface flow

Lateral subsurface flow from the second-growth forest started earlier,
ran later, and generated nearly twice the total runoff compared to the
old growth forest (Fig 1a, Table 2). Concentrations of DOC and TDN
exported in lateral subsurface flow from the old-growth forest were 5
and 3 times higher than those from the second-growth forest and C:N
(DOC/TDN) was also higher in old-growth exports (Table 2, Fig 1b and c),
likely resulting from direct leaching of O-horizon and transport through
shallow, subsurface flowpaths. Peak DOC and TDN concentrations for the
old-growth forest export occurred during the mid-May peak in subsurface
runoff and then declined (Fig 1). In contrast, the DOC and TDN
concentrations in lateral subsurface flow exported from the
second-growth hillslope were uniformly low throughout the flow period.
Annual subsurface flux of C and N were 10 and 8 times higher from the
old-growth hillslope (Table 2).

Characteristics of DOM exported in hillslope lateral subsurface flow

Indices of aromaticity (SUVA\textsubscript{254}) of old-growth DOM
export were 14\% higher (3.46 vs. 3.00) and humification index (HIX) was
nearly twice that of DOM exported from the second-growth forest (12.24
vs. 6.63). The DOM exported from the old-growth hillslope had a mean
fluorescence index (FI) of 1.14 compared to 1.52 from the second-growth
hillslope. Fluorescence regional integration modelling (FRI) confirms
the patterns shown by the EEMs indices (Fig 2). FDOM corresponding to
amino acids (RI and RII) and microbial exudates (RIV) were higher in
exports from the second-growth hillslope. Conversely, plant-derived
fulvic- and humic-like hydrophobic organic acids (RIII and RV) were
higher in exports from the old-growth hillslope.

Indices of FDOM chemical character (FI and HIX) were similar in exports
from the two hillslopes at the onset of subsurface flow but diverged
over time (Fig 3a and 3b). Across the subsurface flow season, FI
decreased by half the possible range in the old-growth hillslope. The
proportion of complex organic acids in FDOM exports (based on HIX),
increased 3-fold during the flow period. In contrast, the chemical
character of FDOM exported from the second-growth hillslope remained
relatively uniform (Fig 3a and 3b).

Reactivity of litter DOM inputs and lateral subsurface flow DOM exports

Patterns of biological reactivity in DOM inputs in litter leachate
between hillslopes matched those of DOM exports in subsurface flow. DOM
inputs and exports on the second-growth hillslope had dissolved oxygen
consumption rates more than twice those from old-growth, reflective of
higher DOM reactivity (Fig 4). Similarly, the average respiratory
quotient of DOM inputs from second-growth was 5 times higher than inputs
to old-growth DOM (1.24, compared to 0.18 for old-growth, Table 2).

\textbf{Discussion}

Forest recovery following timber harvesting and wildfire often shifts
mixed-species subalpine forests to pine-dominant stands (Collins et al.,
2011), and in our study, this transition from old-growth to
second-growth forest decreased hillslope DOM export by 89\% (Tables 1
and 2, Fig 1). The amount of C contained within the O horizon determines
the amount of DOM available for microbial processing and subsurface
transport (Lee, Park, \& Matzner, 2018; Nave, Vance, Swanston, \&
Curtis, 2010). As seen elsewhere we found higher O-horizon C stock, DOC
concentrations and annual export in the old-growth compared to the
second-growth hillslopes (Chatterjee, Vance, Pendall, \& Stahl, 2008;
Chatterjee, Vance, \& Tinker, 2009). In spite of less C contained in
mineral soils of old-growth forests, the higher C exported from them
underscores the importance of subsurface transport of C from soil O
horizons. Our findings suggest that land cover shifts following forest
disturbance (McDowell et al., 2020) and subsequent harvesting is likely
to decrease the amount of C exported to headwater streams.

The chemical character and biological reactivity of DOM inputs from
organic horizons also responded to the land cover change (Supp. Info:
T1, Fig 4). The transition from old- to second-growth forest induced a
change from recalcitrant DOM inputs to more reactive non-humic and
labile compounds such as proteins, small polyphenolics, and
carbohydrates released by pine litter (Beggs \& Summer, 2011; Berg \&
McClaugherty, 2014; Reckhow, Singer, Malcom, 1990; Yavitt \& Fahey,
1984). The higher respiratory quotient (RQ,
\selectlanguage{greek}Δ\selectlanguage{english}CO\textsubscript{2}/\selectlanguage{greek}Δ\selectlanguage{english}O\textsubscript{2}) and dissolved oxygen
consumption rate of input DOM in the second-growth forest reflect this
change in chemical composition and bioavailability (Fig 4) and suggest
that labile proteins or carbohydrates are more readily utilized than
less reactive components common to old-growth DOM (Berggren \& Del
Giorgio, 2012). As opposed to the thin pine needle litter of the
second-growth pine stand, the deep, stratified, O horizon of the
old-growth forests generate less reactive DOM (Table 1).

The forest cover changes altered the reactivity of subsurface DOM
exports. Pine litter produces biologically reactive DOM (Fig 4) that is
subject to microbial transformation as it infiltrates into soils and
moves along subsurface flowpaths (Fig 2 and 3). The greater prevalence
of microbially processed DOM we found in second-growth hillslope exports
agreed with numerous other studies of DOM composition and export after
post-harvest and subsequent forest change (Cawley et al., 2014; Lee \&
Lajtha, 2016; Williams et al., 2010; Yamashita, Kloeppel, Knoepp,
Zausen, \& Jaffe, 2011). In contrast, the DOM exported from old-growth
forest was less biologically reactive (Fig 3b, Fig 4a), consistent with
fluorescence indicators of DOM recalcitrance and complexity (Fig 3a and
3b) and respiratory quotients of litter leachate (Supp. Info: T1, Fig.
4).

We found that DOM reactivity changed in distinct ways in the two forest
types as it moved along subsurface flowpaths (Fig 4). The thick O
horizons in the old-growth forest released more C, though its
recalcitrant, aromatic composition limited the degree of subsurface DOM
processing (Fig 4). In contrast, the more reactive DOM of the
second-growth forest changed substantially downslope. Both our FRI
modelling and earlier work (Cordova et al., 2018; Yano, Lajtha, Sollins,
\& Caldwell, 2005) demonstrate the conversion of labile DOM from litter
into microbial biomass and release of increasingly labile compounds
downslope (Fig 2). The highly reactive nature of second-growth DOM
inputs may also stimulate turnover of C stored in mineral soil (Evans,
Pierson, \& Lajtha, 2020; Kuzyakov, 2010) and agrees with studies that
indicate that microbially processed C may contribute to vertical C
movement from surface litter into the mineral soil profile (Cordova et
al., 2018; Cotrufo, Wallenstein, Boot, Denef, \& Paul, 2013).

The composition of DOM exported from the old-growth hillslope shifts
over the course of the snowmelt season (Fig 3b and c), with a decline in
relatively labile byproducts of microbial processing and an increase in
recalcitrant compounds. This pattern suggests the presence of distinct
DOM sources in this forest type. The DOM transported early in the
snowmelt season may be comprised of labile metabolic byproducts that
accumulated during overwinter microbial transformation of recalcitrant
organic compounds from old-growth litter (Schmidt et al., 2007). After
this initial flush, export DOM may originate from unprocessed compounds
leached directly from the recalcitrant O horizon (Fig 3b). In contrast,
the DOM exported from the second-growth forests was consistently labile
(Fig 3c). This uniform compositional pool may result from the absence of
a recalcitrant source of litter inputs and the continual turnover of C
between labile litter and microbial biomass.

\textbf{Conclusion}

Forested headwater streams drain much of the land area in North America
(Colvin et al., 2019) and are a major contributor of C to the atmosphere
(Cavallaro et al., 2018). Regeneration of pine stands following harvest
of old-growth forests is a common land cover change in the western
United States (Collins et al., 2011) that will have lasting consequences
for the quantity and composition of DOM exported from forest hillslopes
and watershed C cycling. We found that the DOM exported from
second-growth hillslopes was five times more reactive than that from
old-growth, though the total amount of C released was ten times lower.
As such, export of bioavailable C from second-growth was roughly half of
that released from old-growth forest, reducing the amount of DOM
available to stream microbes. The slow recovery of these forests
reinforces the persistent effects of land cover change on DOM
composition and reactivity in headwater ecosystems.

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\textbf{Table 1.} Soil horizon mass, carbon (C) and nitrogen (N) pools,
C and N concentrations, and C:N ratios in organic and mineral (0-10cm)
soil horizons on adjacent old-growth and second-growth forest hillslopes
at the Fraser Experimental Forest, CO. Values are means with standard
errors (n=10 per hillslope type). Within each depth, * and ** denote
differences at p\textless{}0.01 and \textless{}0.001 using a Student's
parametric t-test.

\textbf{Table 2:} Discharge, dissolved organic carbon (DOC), total
dissolved nitrogen (TDN) concentrations and annual fluxes in subsurface
flow from adjacent old-growth and second-growth hillslopes at the Fraser
Experimental Forest, CO. Values are means with standard errors for the
2016, 2017, and 2018 snowmelt seasons. Sample numbers for DOC and TDN
were n=36 for the old-growth and n=48 for second-growth. Differences
between hillslope treatments are noted by ** at p\textless{}0.001 using
a Welch-Satterthwaite non-parametric t-test.

\textbf{Figure 1.} Average daily subsurface flow (A) from adjacent
old-growth and second-growth hillslopes at the Fraser Experimental
Forest for 2016, 2017 and 2018. Dissolved organic carbon (B) and total
dissolved nitrogen (C) in subsurface flow (old-growth n = 36;
second-growth n = 48).

\textbf{Figure 2.} Components of fluorescing DOM (FDOM) in subsurface
flow differentiated by fluorescence regional integration (FRI) modeling.
Values are means from 2016, 2017 and 2018 for adjacent old-growth (n=23)
and second-growth (n=35) hillslopes. Differences between FRI regions
(RI-RV) were identified on logit transformed data at * and ** at
p\textless{}0.01 and 0.001 using a Welch-Satterthwaite non-parametric
t-test.

\textbf{Figure 3.} DOM quality and reactivity in subsurface leachate for
adjacent old-growth and second-growth hillslopes. DOM quality examined
through fluorescence index (A), and humification index (B) for samples
collected during 2016, 2017, and 2018 flow periods (n=23 for second
growth and n=35 for old-growth). Biological oxygen demand assays of DOM
reactivity report oxygen consumption during the 2018 flow period (C).

\textbf{Figure 4.} Reactivity measured as oxygen consumption rates for
DOM extracted from O-horizon litter inputs and subsurface flow exports
(n=10 per each hillslope treatment with standard error bars). The dashed
line shows oxygen consumption for a sample of the native microbial
culture without hillslope DOM added (experimental control). Differences
between hillslope treatments are noted by ** at p \textless{}0.001 using
a Student's parametric t-test.\selectlanguage{english}
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